U.S. patent application number 16/755502 was filed with the patent office on 2021-05-06 for heterobifunctional compounds with improved specificity.
This patent application is currently assigned to DANA-FARBER CANCER INSTITUTE, INC.. The applicant listed for this patent is DANA-FARBER CANCER INSTITUTE, INC.. Invention is credited to Eric S. FISCHER, Nathanael S. GRAY, Brian GROENDYKE, Zhixiang HE, Radoslaw P. NOWAK, Tinghu ZHANG.
Application Number | 20210130368 16/755502 |
Document ID | / |
Family ID | 1000005355151 |
Filed Date | 2021-05-06 |
United States Patent
Application |
20210130368 |
Kind Code |
A1 |
NOWAK; Radoslaw P. ; et
al. |
May 6, 2021 |
HETEROBIFUNCTIONAL COMPOUNDS WITH IMPROVED SPECIFICITY
Abstract
Disclosed are heterobifunctional compounds that effectuate
selective degradation of a target protein, and which include a
targeting ligand that binds a target protein and at least one other
protein, a ligand that binds an E3 ubiquitin ligase or a component
of E3 ubiquitin ligase, and a specificity modulating linker that
links the first ligand and the second ligand. Pharmaceutical
compositions containing the compounds, and methods of using and
making the compounds are also disclosed.
Inventors: |
NOWAK; Radoslaw P.; (Boston,
MA) ; FISCHER; Eric S.; (Newton, MA) ; GRAY;
Nathanael S.; (Boston, MA) ; ZHANG; Tinghu;
(Brookline, MA) ; HE; Zhixiang; (Brookline,
MA) ; GROENDYKE; Brian; (Chestnut Hill, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DANA-FARBER CANCER INSTITUTE, INC. |
Boston |
MA |
US |
|
|
Assignee: |
DANA-FARBER CANCER INSTITUTE,
INC.
Boston
MA
|
Family ID: |
1000005355151 |
Appl. No.: |
16/755502 |
Filed: |
October 19, 2018 |
PCT Filed: |
October 19, 2018 |
PCT NO: |
PCT/US18/56680 |
371 Date: |
April 10, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62575156 |
Oct 20, 2017 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
C07D 401/04 20130101; C07D 487/04 20130101; C07D 401/12 20130101;
A61K 47/55 20170801; C07D 211/56 20130101; C07D 495/14
20130101 |
International
Class: |
C07D 495/14 20060101
C07D495/14; C07D 487/04 20060101 C07D487/04; C07D 401/04 20060101
C07D401/04; C07D 401/12 20060101 C07D401/12; C07D 211/56 20060101
C07D211/56; A61K 47/55 20060101 A61K047/55; A61P 35/00 20060101
A61P035/00 |
Goverment Interests
GOVERNMENT LICENSE RIGHTS
[0002] This invention was made with government support under grant
number R01 CA214608 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Claims
1. A heterobifunctional compound or pharmaceutically acceptable
salt or stereoisomer of Formula (I): ##STR00091## wherein n is 0 or
1; m is 0 or 1; p is 0 or 1; and R.sub.1 is an ether, an alkyl
ether, an alkyl amine, C.sub.1 alkyl, C.sub.2 alkyl, C.sub.3 alkyl,
C.sub.4 alkyl, C.sub.5 alkyl, C.sub.6 alkyl, or a 5- or 6-member
cyclic group; wherein L.sub.1 binds a target protein and at least
one protein; and wherein L.sub.2 binds an E3 ubiquitin ligase or a
component of an E3 ubiquitin ligase, or a pharmaceutically
acceptable salt, ester or stereoisomer thereof, wherein the
compound selectively degrades the target protein relative to the at
least one other protein to which L.sub.1 binds.
2. The heterobifunctional compound of claim 1, wherein R.sub.1 is a
polyethylene glycol chain ranging from 1 to 2 ethylene glycol
units.
3. The heterobifunctional compound of claim 1, wherein the linker
is selected from the structures: ##STR00092##
4. The heterobifunctional compound of claim 1, wherein the at least
one other protein to which L.sub.1 binds is homologous to the
target protein.
5. The heterobifunctional compound of claim 4, wherein the target
protein to which L.sub.1 binds is BRD4 and the at least one other
protein to which L.sub.1 binds is a bromo extra-terminal domain
(BET) protein, wherein the targeting ligand L.sub.1 binds the
target protein with greater affinity than the at least one other
BET protein, the targeting ligand L.sub.1 binds the target protein
with lesser affinity than the at least one other BET protein, or
the targeting ligand L.sub.1 binds the target protein with
substantially equal affinity than the at least one other BET
protein.
6. (canceled)
7. (canceled)
8. (canceled)
9. The heterobifunctional compound of claim 5, wherein L.sub.1 also
binds BRD2 and BRD3.
10. The heterobifunctional compound of claim 9, wherein L.sub.1 is
JQ1, ##STR00093## or an analog thereof, and the target protein is
BRD4.
11. The heterobifunctional compound of claim 10, L.sub.1 is
represented by structure C.sub.3: ##STR00094## Structure 2, wherein
##STR00095## wherein R.sub.3 is methyl or ##STR00096## R.sub.2 is
##STR00097## and R.sub.4 is ##STR00098## wherein L.sub.1 is
selected from the structures: ##STR00099## ##STR00100##
12. (canceled)
13. The heterobifunctional compound of claim 1, wherein L.sub.2
binds cereblon or VHL.
14. The heterobifunctional compound of claim 13, wherein L.sub.2
binds cereblon and is selected from the structures: ##STR00101##
##STR00102##
15. (canceled)
16. The heterobifunctional compound of claim 1, wherein the
bifunctional compound of formula (I) is represented by any of the
following structures: ##STR00103## ##STR00104## ##STR00105##
##STR00106## and pharmaceutically acceptable salts and
stereoisomers thereof.
17. A pharmaceutical composition, comprising a therapeutically
effective amount of the heterobifunctional compound or
pharmaceutically acceptable salt or stereoisomer of claim 1, and a
pharmaceutically acceptable carrier.
18. A method of treating a disease or disorder mediated by
dysfunctional or dysregulated proteins activities comprising
administering to a subject in the need thereof the
heterobifunctional compound or pharmaceutically acceptable salt or
stereoisomer of claim 1.
19. The method of claim 18, wherein the subject has cancer.
20. The method of claim 19, wherein the subject has a NUT midline
carcinoma.
21. A method of selectively degrading a target protein that is a
member of a family of homologous proteins, the method comprising
contacting a cell with the heterobifunctional compound or
pharmaceutically acceptable salt or stereoisomer of claim 1 under
conditions and for a period of time sufficient to result in
selective degradation of the target protein.
22. The method of claim 21, wherein the cell is a human cell or a
mouse cell.
23. The method of claim 21, wherein the cell is HEK293, HEK293T,
MM.1S, MOLM-13, MV4:11, or a THP-1 cell.
Description
RELATED APPLICATION
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn. 119(e) to U.S. Provisional Application No. 62/575,156
filed Oct. 20, 2017, which is incorporated herein by reference in
its entirety.
FIELD OF THE INVENTION
[0003] The present invention generally relates to
heterobifunctional small molecule compounds (also known as
PROTACs), and more specifically to heterobifunctional small
molecule degraders that can degrade a target protein with high
levels of specificity.
BACKGROUND OF THE INVENTION
[0004] While long sought after, rational design of synthetic
chemical matter that is capable of inducing selective protein
dimerization has remained challenging. Significant progress has
recently been made towards chemically induced targeted protein
degradation using heterobifunctional compounds also known as
degraders or PROTACs (PROteolysis-TArgeting Chimeras) (Bondeson et
al., 2015; Buckley et al., 2015; Gadd et al., 2017; Gustafson et
al., 2015; Kenten & Roberts, 2001; J. Lu et al., 2015; Sakamoto
et al., 2001; Winter et al., 2015). Targeted protein degradation
refers to small molecule induced ubiquitination and degradation of
disease targets, in which the small molecule simultaneously
recruits both a ubiquitin E3 ligase and the target protein into
close proximity of each other which leads to ubiquitination of the
target protein. Clinical proof of concept for targeted protein
degradation is provided by the recent discovery that the potent
anti-cancer drugs thalidomide, lenalidomide and pomalidomide
(collectively known as IMiDs) exert their thersapeutic effects
through induced degradation of key efficacy targets, such as IKZF1,
IKZF3, ZFP91, or casein kinase 1 alpha (Ck1.alpha.) (An et al.,
2017; Kronke et al., 2015; G. Petzold, Fischer, & Thoma, 2016).
IMiDs bind cereblon (CRBN), the substrate receptor of the E3
ubiquitin ligase, and act by redirecting the activity of the
CRL4.sup.CRBN ligase to ubiquitinate the proteins targeted for
degradation. (Chamberlain et al., 2014; Fischer et al., 2014; Ito
et al., 2010); G. Petzold et al., 2016).
[0005] PROTACs (or degraders) typically contain an E3 ligase
binding scaffold (E3-moiety), which is often an analogue of
thalidomide (which bind to the E3 ubiquinase known as cereblon), or
a ligand to the von Hippel-Lindau tumor suppressor (VHL) protein
(Buckley et al., 2012), which is attached via a linker to another
small molecule (target-moiety) that binds a target protein of
interest (FIG. 1A and FIGS. 7A and B). Recruitment of the target
protein to the E3 ubiquitin ligase facilitates ubiquitination and
subsequent degradation of the target protein (Raina & Crews,
2017). This principle has been successfully applied to several
targets including the Bromodomain and Extra Terminal (BET) family
(BRD2, BRD3, BRD4), RIPK2, BCR-ABL, FKBP12, BRD9, and ERRa Thus,
PROTACs constitute a promising new pharmacologic modality and is
being widely explored in chemical biology and drug discovery.
(Bondeson et al., 2015; Lai et al., 2016; J. Lu et al., 2015; Raina
et al., 2016; Remillard et al., 2017; Toure & Crews, 2016;
Winter et al., 2015).
[0006] However, among other issues, the selectivity of degraders
for target proteins can be unpredictable. For example, MZ1 which is
a PROTAC that contains a VHL-ligand linked to the BRD4 ligand JQ1,
showed complexation not only with the second bromodomain of BRD4
(BRD4.sub.BD2) but also with the second bromodomains of the
homologous BET proteins BRD2 and BRD3. It remains to be seen if
PROTACs that target BRD4 for degradation via recruitment of
cereblon exhibit similar binding profiles. Based upon these current
limitations, there remains a need for heterobifunctional compounds
(PROTACs) that can selectively degrade a target protein to the
substantial exclusion of other homologous proteins.
SUMMARY OF THE INVENTION
[0007] A first aspect of the present invention provides a
heterobifunctional compound that selectively degrades a target
protein. The heterobifunctional compounds of the present invention
are represented by Formula (I):
##STR00001##
wherein n is 0 or 1; m is 0 or 1; p is 0 or 1; and R.sub.1 is an
ether, an alkyl ether, an alkyl amine, C.sub.1 alkyl, C.sub.2
alkyl, C.sub.3 alkyl, C.sub.4 alkyl, C.sub.5 alkyl, C.sub.6 alkyl,
or a 5- or 6-member cyclic group; wherein L.sub.1 binds a target
protein and at least one other protein; and wherein L.sub.2 binds
an E3 ubiquitin ligase, or a pharmaceutically acceptable salt,
ester or stereoisomer thereof. In some embodiments, the at least
one other protein to which L.sub.1 binds has a high sequence
identity with the target protein. In some embodiments, the at least
one other protein to which L.sub.1 binds is homologous to the
target protein.
[0008] The heterobifunctional compounds enable selective
ubiquitin-mediated degradation of the target protein relative to
the at least one other protein to which L.sub.1 binds, regardless
of whether L.sub.1 binds to the at least one other protein with
equal, greater or lesser affinity. Thus, the inventive
heterobifunctional compounds may be useful in selectively degrading
a specific protein that is implicated in a disease or condition. In
some embodiments, the at least one other protein to which L.sub.1
binds is left substantially non-degraded.
[0009] In some embodiments, the heterobifunctional compound
includes a targeting ligand L.sub.1 that binds bromodomain protein
BRD4 (e.g., to the first and/or second bromodomains of BRD4) and to
at least one other protein which is BRD3 and/or BDR2 with similar
affinity, but enables the selective degradation of BRD4. In some
embodiments, the targeting ligand binds the bromodomain proteins
BRD2, BRD3, and BRD4 with substantially equal affinity, yet the
heterobifunctional compound selectively degrades BRD4. In some
embodiments, the targeting ligand is JQ1 or an analog thereof.
[0010] A second aspect of the invention provides a pharmaceutical
composition comprising an effective amount of the
heterobifunctional compound, or a pharmaceutically acceptable salt,
ester or stereoisomer thereof.
[0011] In various aspects, the invention provides a method of
treating a subject having a disease or disorder mediated by
dysfunctional or dysregulated protein function, comprising
administering to a subject in need thereof the heterobifunctional
compound which targets the dysfunctional or dysregulated
protein.
[0012] In various aspects, the invention provides a method of
selectively degrading a target protein by contacting a cell with
the heterobifunctional compound under conditions and for a period
of time sufficient to result in selective degradation of the target
protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1A is an image that shows the chemical structure of
dBET23 with the target-moiety in red, the linker in black and
green, and the E3-moiety in blue.
[0014] FIG. 1B is an image that shows a cartoon representation of
DDB1AB-CRBN-dBET23-BRD4.sub.BD1: DDB1 highlighting domains BPA
(red), BPC (orange) and DDB1-CTD (grey); CRBN with domains NTD
(blue), HBD (cyan) and CTD (green); and BRD4.sub.BD1 (magenta). The
Zn.sup.2+-ion is shown as a grey sphere and dBET23 as sticks
representation in yellow. The F.sub.O-F.sub.C map is shown as green
mesh for dBET23 contoured at 3.0.sigma..
[0015] FIG. 1C is an image that shows superposition of
DDB1.DELTA.B-CRBN-dBET23-BRD4.sub.BD1 with human CRBN bound to
lenalidomide (PDB: 4tz4) and BRD4.sub.BD1 bound to JQ1-(S) (PDB:
3mxf). Surface representation for CRBN and BRD4.sub.BD1 are shown
in grey and magenta, respectively. dBET23 is shown in yellow, JQ1
in green, and thalidomide in cyan.
[0016] FIG. 1D is an image that shows side-chain interactions
between BRD4.sub.BD1, CRBN, and dBET23. Dashed lines indicate
hydrogen bonds. Residues of BRD4.sub.BD1 mutated in this study are
highlighted in cyan.
[0017] FIG. 2A-FIG. 2F show data demonstrating that dBET mediated
BRD4 recruitment is governed by negative cooperativity. All data in
FIG. 2A, FIG. 2C, and FIG. 2D represent biological replicates
presented as means.+-.s.d. (n=3).
[0018] FIG. 2A is a bar graph that shows TR-FRET data where dBET23
is titrated to DDB1.DELTA.B-CRBN.sub.SPY-BODIPY,
Terbium-Streptavidin and various BRD4.sub.BD1-biotin wild type and
mutant proteins. The mean peak heights for dose response curves of
three independent replicates are shown as bar charts.
[0019] FIG. 2B is an image that shows surface representation of
CRBN highlighting the residues involved in dBET23 mediated
BRD4.sub.BD1 binding in orange.
[0020] FIG. 2C is a graph that shows competitive binding assay for
dBET1 binding to DDB1.DELTA.B-CRBN. Increasing concentrations of
dBET1 titrated to preformed
DDB1.DELTA.B-CRBN-lenalidomide.sub.Atto565 complex in presence or
absence of BRD4.sub.BD1 or BRD4.sub.BD2 are shown.
[0021] FIG. 2D-FIG. 2F are graphs that show similar competitive
assays for dBET6, dBET23 and dBET57, respectively.
[0022] FIG. 3A-FIG. 3F are graphs that show quantitative assessment
of cellular degradation for BRD4.sub.BD1 and BRD4.sub.BD2. Data in
FIG. 3A-FIG. 3F represent four biological replicates analyzed in
technical duplicates with 5000 cells each, and presented as the
means.+-.s.d.
[0023] FIG. 3A-FIG. 3C are graphs that show quantitative assessment
of cellular degradation using a BRD4.sub.BD1-EGFP reporter assay.
Cells stably expressing BRD4.sub.BD1-EGFP and mCherry were treated
with increasing concentrations of lenalidomide, dBET1, dBET6,
dBET23, dBET55, dBET57, dBET70, and MZ1 and the EGFP and mCherry
signals followed using flow cytometry analysis.
[0024] FIG. 3D-FIG. 3F are graphs that show quantitative assessment
of cellular degradation using a BRD4.sub.BD2-EGFP reporter assay.
Cells stably expressing BRD4.sub.BD2-EGFP and mCherry were treated
with increasing concentrations of dBET1, dBET6, dBET23, dBET55,
dBET57, dBET70, MZ1 and lenalidomide. EGFP and mCherry signals were
measured using flow cytometry analysis.
[0025] FIG. 4A-FIG. 4H show data demonstrating plasticity of
CRBN-substrate interactions.
[0026] FIG. 4A is a bar graph that shows TR-FRET data where dBET23
is titrated to BRD4.sub.BD1-SPYCATCHER-BODIPY, Terbium-antiHis
antibody and various His6-DDB1.DELTA.B-CRBN wild type and
His6-DDB1-CRBN mutant proteins. The mean peak heights for dose
response curves of three independent replicates are shown as bar
charts.
[0027] FIG. 4B is a bar graph that shows TR-FRET data where dBET23
is titrated to DDB1.DELTA.B-CRBN.sub.SPYCATCHER-BODIPY,
Terbium-Streptavidin and various BRD4.sub.BD1-biotin wild type and
mutant proteins. The mean peak heights for dose response curves of
three independent replicates are shown as bar charts.
[0028] FIG. 4C is a bar graph that shows TR-FRET data where dBET57
is titrated to BRD4.sub.BD1-SPYCATCHER-BODIPY, Terbium-antiHis
antibody and various His6-DDB1.DELTA.B-CRBN wild type and
His6-DDB1-CRBN mutant proteins.
[0029] FIG. 4D is a bar graph that shows TR-FRET data where dBET57
is titrated to DDB1.DELTA.B-CRBN.sub.SPYCATCHER-BODIPY,
Terbium-Streptavidin and various BRD4.sub.BD1-biotin wild type and
mutant proteins. Data in FIG. 4A-FIG. 4D represent biological
replicates presented as means.+-.s.d. (n=3).
[0030] FIG. 4E is an image that shows the chemical structure of
dBET57 with the target-moiety in red, the linker in black and
green, and the E3-moiety in blue.
[0031] FIG. 4F is an image that shows a cartoon representation of
DDB1.DELTA.B-CRBN-dBET57-BRD4.sub.BD1: DDB1 highlighting domains
BPA (red), BPC (orange) and DDB1-CTD (grey); CRBN with domains NTD
(blue), HBD (cyan) and CTD (green); BRD4.sub.BD1 (magenta). The
Zn.sup.2+-ion is drawn as a grey sphere. dBET57 was not modelled in
this structure but instead superpositions of lenalidomide (from
pdb: 5fqd) and JQ1 (from pdb: 3mxf) are shown in yellow sticks.
[0032] FIG. 4G is an image that shows superposition of CRBN and
BRD4.sub.BD1 for the dBET23 and dBET57 containing complexes.
Superposition was carried out over the CRBN-CTD (residues
320-400).
[0033] FIG. 4H is an image that shows surface representation of
CRBN highlighting the BRD4.sub.BD1 interacting residues for the
dBET57 mediated recruitment in orange.
[0034] FIG. 5A-FIG. 5C show in silico docking to predict binding
modes.
[0035] FIG. 5A is an image of an interface RMSD that shows
symmetric docking energy landscape for the binding of BRD4.sub.BD1
to a CRBN-lenalidomide complex. The two low energy decoys that
exhibit a conformation compatible with dBET binding are indicated
by bold numbers. The symmetric docking energy landscape for local
perturbation docking experiments on decoy 12662 compatible with
dBET mediated binding is shown as insert.
[0036] FIG. 5B is an image that shows superposition of the
DDB1.DELTA.B-CRBN-dBET23-BRD4.sub.BD1 structure and the top
solution from local perturbation of decoy 12662.
[0037] FIG. 5C is an image that shows cartoon representations of
three representative clusters from the global docking run.
[0038] FIG. 6A-FIG. 6H show data demonstrating degradation of BET
family proteins by certain heterobifunctional small molecule
degraders.
[0039] FIG. 6A is an image that shows a cartoon representation of
structures from cluster 19, and close-up view highlighting the
proximity of the JQ1 analog and lenalidomide that provided the
rationale for synthesizing the heterobifunctional small molecule
degrader ZXH-03-26, which is shown in FIG. 6B.
[0040] FIG. 6C is a graph that shows quantitative assessment of
cellular degradation using a EGFP/mCherry reporter assay. Cells
stably expressing BRD4.sub.BD1-EGFP (or constructs harbouring
BRD2.sub.BD1, BRD2.sub.BD2, BRD3.sub.BD1, BRD3.sub.BD2,
BRD4.sub.BD2) and mCherry were treated with increasing
concentrations of ZXH-03-26 and the EGFP and mCherry signals
followed using flow cytometry analysis.
[0041] FIG. 6D-FIG. 6F are graphs that show quantitative assessment
of cellular degradation using a EGFP/mCherry reporter assay. Cells
stably expressing BRD4.sub.BD1-EGFP (or constructs harbouring
BRD2.sub.BD1, BRD2.sub.BD2, BRD3.sub.BD1, BRD3.sub.BD2,
BRD4.sub.BD2) and mCherry were treated with increasing
concentrations dBET6 (FIG. 6D), MZ1 (FIG. 6E), and dBET57 (FIG.
6F).
[0042] FIG. 6G are immunoblots that demonstrate cellular
degradation of endogenous BRD4 in HEK293T cells that were treated
with increasing concentrations of ZXH-03-26 or dBET6 for 5
hours.
[0043] FIG. 6H s are immunoblots that show degradation of BRD2 and
BRD3 by compounds dDEBT6 and ZXH-03-26.
[0044] FIG. 7A is an image that shows a schematic representation of
the heterobifunctional ligand (PROTAC/degrader) mediated
degradation.
[0045] FIG. 7B is an image that shows chemical structures,
molecular weight and CLogP for the heterobifunctional small
molecule degraders (BET inhibitor JQ1-(S) coloured in red,
thalidomide moiety coloured in blue and the linker in black and
green).
[0046] FIG. 7C is an image that shows multiple sequence alignment
of BD1 and BD2 from different BET bromodomain paralogs.
[0047] FIG. 7D is an image that shows multiple sequence alignment
of BD1 and BD2 from human BRD4.
[0048] FIG. 7E is an image that shows domain architecture of BDR4
(A and B-DNA binding motifs; ET--external domain;
SEED--Ser/Glu/Asp-rich region; CTM--C-terminal domain).
[0049] FIG. 8A is an image that shows a cartoon representation of
DDB1.DELTA.B-CRBN-dBET6-BRD4.sub.BD1. The F.sub.O-F.sub.C map is
shown as green mesh for dBET6 contoured at 4.06.
[0050] FIG. 8B is an image that shows a cartoon representation of
DDB1.DELTA.B-CRBN-dBET70-BRD4.sub.BD1. The F.sub.O-F.sub.C map is
shown as green mesh for dBET70 contoured at 4.0.sigma..
[0051] FIG. 8C is an image that shows a cartoon representation of
DDB1.DELTA.B-CRBN-dBET55-BRD4.sub.BD1/D145A. The F.sub.O-F.sub.C
map is shown as green mesh contoured at 3.0.sigma.. In FIGS. 8A-C,
DDB1 is shown in grey, CRBN in blue, and BRD4.sub.BD1 (wildtype and
mutant) in magenta.
[0052] FIG. 8D-FIG. 8J are tables that show TR-FRET data underlying
bar charts shown in FIG. 2A, FIG. 4A-FIGS. 4D and 11D-L. The
TR-FRET data in FIG. 8D-FIG. 8J represent biological replicates
presented as means.+-.s.d. (n=3).
[0053] FIG. 9A-FIG. 9H show data demonstrating negative
cooperativity governing CRBN-dBET-BRD4 interactions.
[0054] FIG. 9A is an image that shows a schematic of fluorescence
polarization based CRBN binding assay. Atto565-Lenalidomide
fluorophore is displaced by PROTAC bound BRD4.sub.BD1/2.
[0055] FIG. 9B is a graph that shows fluorescence polarization
competitive binding assay for dBET55 binding to DDB1.DELTA.B-CRBN.
Increasing concentrations of dBET55 titrated to preformed
DDB1.DELTA.B-CRBN-lenalidomide.sub.Atto565 complex in presence or
absence of BRD4.sub.BD1 or BRD4.sub.BD2.
[0056] FIG. 9C-FIG. 9G are graphs that show fluorescence
polarization competitive binding assay for dBET1, dBET6, dBET23,
dBET55, and dBET57, respectively, to DDB1.DELTA.B-CRBN with
increasing concentrations of dBETs titrated to preformed
DDB1.DELTA.B-CRBN-lenalidomide.sub.Atto565 complex in presence or
absence of BRD4.sub.BD1 or BRD4.sub.BD2 at concentrations of 1
.mu.M, 5 .mu.M, and 20 .mu.M. The data at 5 .mu.M BRD4.sub.BD1/2
was replotted for FIGS. 2C-F and FIG. 9B.
[0057] FIG. 9H is a table that shows summary of apparent
cooperativity factors .alpha..sub.app.
[0058] FIG. 10A-FIG. 10L are graphs that show quantitative
assessment of cellular degradation of
BRD4.sub.BD1-EGFP/BRD4.sub.BD2-EGFP and IKZF1.DELTA.-EGFP by
lenalidomide, dBET1, dBET6, dBET23, dBET55, dBET57, dBET70, dBET72,
MZ1, ZXH-2-42, ZXH-2-43, and ZXH-2-45, respectively, using flow
cytometry analysis. Cells stably expressing
BRD4.sub.BD1-EGFP/BRD4.sub.BD2-EGFP or IKZF1.DELTA.-EGFP with a
mCherry reporter were treated with increasing concentrations of the
heterobifunctional small molecule degraders with the EGFP and
mCherry signals quantified using flow cytometry analysis.
[0059] FIG. 11A-FIG. 11I show plasticity of CRBN-substrate
interactions.
[0060] FIG. 11A is an image that shows the different surfaces CRBN
utilizes to interact with a variety with neo-substrates as
illustrated by the superposition of
DDB1.DELTA.B-CRBN-dBET23-BRD4.sub.BD1,
DDB1.DELTA.B-CRBN-lenalidomide-Ck1.alpha. (PDB entry 5fqd), and
DDB1-CRBN-CC885-GSPT1 (PDB entry 5hxb). A close-up of the common
hydrophobic interface between GSPT1-CRBN-NTD and
BRD4.sub.BD1-CRBN-NTD is shown in the top right box.
[0061] FIG. 11B is a graph that shows a competitive binding assay
where titrating BRD4.sub.BD1 or BRD4.sub.BD2 into a preformed
complex of DDB1-CRBN-dBET57-IKZF1.DELTA.demonstrated mutually
exclusive binding of BRD4 with neosubstrates such as Ck1.alpha. or
IKZF1/3.
[0062] FIG. 11C is an image that shows a surface representation of
CRBN and BRD4.sub.BD1 of DDB1-CRBN-dBET23-BRD4.sub.BD1 crystal
structure, showing dBET23 as stick representation. The hypothetical
linker path from the acid position on JQ1 is shown with red spheres
indicating the distance of a carbon-carbon bond and illustrating
that the 2-carbon linker of dBET57 would be insufficient to bridge
the gap.
[0063] FIG. 11D is a graph that shows TR-FRET data where dBET6
degrader was titrated to BRD4.sub.BD1SPYCATCHER-BODIPY
Terbium-antiHis antibody, and wild type or various mutants of
His6-DDB1-His6-CRBN complex. The peak height of the dose response
curve for three independent replicates was quantified and is
depicted as bar charts. The TR-FRET data in this figure are
biological replicates presented as means.+-.s.d. (n=3).
[0064] FIG. 11E shows TR-FRET data where dBET6 degrader was
titrated to DDB1.DELTA.B-CRBN.sub.SPYCATCHER-BODIPY,
Terbium-Streptavidin and wild type or mutants of
BRD4.sub.BD1-biotin. The peak height of the dose response curve for
three independent replicates was quantified and is depicted as bar
charts. The TR-FRET data in this figure are biological replicates
presented as means.+-.s.d. (n=3).
[0065] FIG. 11F and FIG. H are graphs that show TR-FRET data where
dBET1 and dBET55, respectively, were titrated to
BRD4.sub.BD1SPYCATCHER-BODIPY Terbium-antiHis antibody, and wild
type or various mutants of His6-DDB1-His6-CRBN complex. The peak
height of the dose response curve for three independent replicates
was quantified and is depicted as bar charts. The TR-FRET data in
this figure are biological replicates presented as means.+-.s.d.
(n=3).
[0066] FIG. 11G and FIG. 11I are graphs that show TR-FRET data
where dBET1 and dBET55, respectively, were titrated to
BRD4.sub.BD1SPYCATCHER-BODIPY Terbium-antiHis antibody, and wild
type or various mutants of His6-DDB1-His6-CRBN complex. The peak
height of the dose response curve for three independent replicates
was quantified and is depicted as bar charts. The TR-FRET data in
this figure are biological replicates presented as means.+-.s.d.
(n=3).
[0067] FIG. 12A-FIG. 12C show experimental validation of
DDB1-CRBN-dBET57-BRD4.sub.BD1 structure.
[0068] FIG. 12A is an image that shows a cartoon representation of
DDB1-CRBN-dBET57-BRD4.sub.BD1 complex with the 2F.sub.O-F.sub.C map
contoured at 1.5.sigma.. Domains are coloured as DDB1-BPA (red),
DDB1-BPC (orange), DDB1-CTD (grey), CRBN-NTD (blue), CRBN-HBD
(cyan), CRBN-CTD (green), and BRD4.sub.BD1 (magenta).
[0069] FIG. 12B is an image that shows anomalous difference map
contoured at 4.sigma. shown in green for data collected at the Zn
peak showing the position of the Zn in the final model. 2
F.sub.O-F.sub.C map is shown as blue mesh.
[0070] FIG. 12C is an image that shows F.sub.O-F.sub.C map
contoured at 3.5.sigma. and shown in green and red, together with 2
F.sub.O-F.sub.C map contoured at 1.5.sigma. and shown in blue.
Positive difference density is observed for the Thalidomide (Thal)
and JQ1 binding sites.
[0071] FIG. 13A-FIG. 13D show in silico docking of
CRBN-lenalidomide-Ck1 complex.
[0072] FIG. 13A is an image of an interface RMSD shows symmetric
docking energy landscape for the binding of Ck1.alpha. to a
CRBN-lenalidomide complex. Symmetric docking energy landscape for
local perturbation docking experiments on a lowest energy decoy
00689 is shown as an insert.
[0073] FIG. 13B is an image that shows superposition of the
DDB1.DELTA.B-CRBN-lenalidomide-Ck1.alpha. structure (PDB: 5fqd) and
the top solution, decoy 0173, from FIG. 13A.
[0074] FIG. 13C is an image of an interface RMSD shows symmetric
energy docking landscape for the binding of Ck1.alpha. to a
CRBN-lenalidomide complex. The conformer parameter file for
lenalidomide was restricted to a conformer not favorable of
Ck1.alpha. binding.
[0075] FIG. 13D is an image that shows superposition of the
DDB1.DELTA.B-CRBN-lenalidomide-Ck1.alpha. structure (PDB: 5fqd) and
the top solution from FIG. 13C.
[0076] FIG. 14A-FIG. 14E show co-degradation of IMiD neo-substrates
such as IKZF1/3.
[0077] FIG. 14A is a graph that shows TR-FRET data where titration
of the indicated molecules to
DDB1.DELTA.B-CRBN.sub.SPYCATCHER-BODIPY, Terbium-streptavidin and
IKZF1.DELTA..sub.biotin. Data in this figure are presented as
means.+-.s.d. (n=3).
[0078] FIG. 14B is a graph that shows quantitative assessment of
cellular degradation of an IKZF1-EGFP reporter using flow cytometry
analysis. Cells stably expressing IKZF1.DELTA.-EGFP and mCherry
were treated with increasing concentrations of the indicated
molecules and the EGFP and mCherry signals followed using flow
cytometry analysis. Data in this figure are presented as
means.+-.s.d. (n=4).
[0079] FIG. 14C is an image that shows a model of a CRBN-IKZF1ZnF2
complex (adapted from Petzold et al., 2016) bound to lenalidomide.
Potential hydrogen bonds are indicated as dashed lines.
[0080] FIG. 14D is a scatter plot that shows the fold changes in
relative abundance comparing dBET23 to DMSO control treatment
(MM.1s) determined using quantitative proteomics. Negative false
discovery rate adjusted P Values are shown on the x-axis and log 2
fold changes on the y-axis. Data shown are three biological
replicates measured in a single 10-plex TMT experiment.
[0081] FIG. 14E is a scatter plot that shows a similar experiment
as FIG. 14D but for dBET70 to DMSO control.
[0082] FIG. 15A-FIG. 15C show selective degradation of BRD4 by
certain heterobifunctional small molecule degraders ZXH-3-147 and
184, as compared to non-selective degradation of BET family
proteins by ZXH-3-27.
[0083] FIG. 15A is a graph that shows selective degradation of BRD4
by ZXH-2-147 using quantitative assessment of cellular degradation
using EGFP/mCherry reporter assay. Cells stably expressing
BRD4.sub.BD1-EGFP (or constructs harbouring BRD2.sub.BD1,
BRD2.sub.BD2, BRD3.sub.BD1, BRD3.sub.BD2, BRD4.sub.BD2) and mCherry
were treated with increasing concentrations of ZXH-02-147 and the
EGFP and mCherry signals followed using flow cytometry
analysis.
[0084] FIG. 15B is a graph that shows selective degradation of BRD4
by ZXH-2-184 using the same quantitative assessment as FIG.
15A.
[0085] FIG. 15C is a graph that shows a lack of selective
degradation of BRD4 by ZXH-3-27 using the same quantitative
assessment as FIG. 15A.
[0086] FIG. 16A-FIG. 16L shows selective degradation of BRD4 by
certain heterobifunctional small molecule degraders.
[0087] FIG. 16A, FIG. 16C, FIG. 16E, FIG. 16G, FIG. 16I, and FIG.
16K show chemical structures of ZXH-3-79, ZXH-3-27, ZXH-2-147,
ZXH-2-184, ZXH-3-26, and ZXH-3-82.
[0088] FIG. 16B, FIG. 16D, FIG. 16F, FIG. 16H, FIG. 16J, and FIG.
16L show degradation of BRD4 by ZXH-3-79, ZXH-3-27, ZXH-2-147,
ZXH-2-184, ZXH-3-26, and ZXH-3-82, respectively, via quantitative
assessment of cellular degradation using EGFP/mCherry reporter
assay. Cells stably expressing BRD4.sub.BD1-EGFP (or constructs
harbouring BRD2.sub.BD1, BRD2.sub.BD2, BRD3.sub.BD1, BRD3.sub.BD2,
BRD4.sub.BD1, BRD4.sub.BD2) and mCherry were treated with
increasing concentrations of ZXH-03-79 and the EGFP and mCherry
signals followed using flow cytometry analysis.
[0089] FIG. 17A-FIG. 171 are bar graphs that show TR-FRET data
illustrating mutational profiles of various heterobifunctional
compounds. TR-FRET data for dBET1 (FIG. 17A), dBET6 (FIG. 17B),
dBET23 (FIG. 17.C), dBET55 (FIG. 17D), dBET57 (FIG. 17E), ZXH-3-26
(FIGS. 17F and H) and dBET70 (FIGS. 17G and I) titrated to
DDB1.DELTA.B-CRBN.sub.SPYCATCHER-BODIPY, Terbium-Streptavidin and
various BRD4.sub.BD1-biotin wild type and mutant proteins are
shown. The mean peak heights for dose response curves of three
independent replicates are shown as bar charts. The TR-FRET data in
FIGS. 17A-I represent biological replicates presented as
means.+-.s.d. (n=3).
[0090] FIG. 18 is bar graph that shows histogram of shortest
pairwise distances found in docking poses between solvent exposed
atoms of JQ1 bound to BRD4.sub.BD1 and Lenalidomide bound to CRBN.
Distances from 10,000 docking poses are shown in black and top 200
poses based on the docking score in gray.
[0091] FIG. 19A-FIG. 19G are graphs that show selective degradation
of bromodomains by BJG-02-119, BSJ-02-174BJG-02-030, ZXH-3-52,
ZXH-3-195, ZXH-3-28, and ZXH-4-28, respectively, via quantitative
assessment of cellular degradation using EGFP/mCherry reporter
assay. Cells stably expressing BRD4.sub.BD1-EGFP (or constructs
harbouring BRD2.sub.BD1, BRD2.sub.BD2, BRD3.sub.BD1, BRD3.sub.BD2,
BRD4.sub.BD1, BRD4.sub.BD2) and mCherry were treated with
increasing concentrations of degrader, incubated for 5 h, and the
EGFP and mCherry signals followed using cellular imaging-based
degradation assay. BSJ-02-119 and BSJ-02-174 n=2, others n=1.
[0092] FIG. 20A-FIG. 20D are graphs that show degradation of
bromodomains by ZXH-3-117, ZXH-2-42, ZXH-2-43, ZXH-2-45. Cells
stably expressing BRD4.sub.BD1-EGFP (or constructs harbouring
BRD2.sub.BD1, BRD2.sub.BD2, BRD3.sub.BD1, BRD3.sub.BD2,
BRD4.sub.BD1, BRD4.sub.BD2) and mCherry were treated with
increasing concentrations of degrader, incubated for 5 h, and the
EGFP and mCherry signals followed using cellular imaging-based
degradation assay, n=1.
DETAILED DESCRIPTION OF THE INVENTION
[0093] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which the subject matter herein belongs. As
used in the specification and the appended claims, unless specified
to the contrary, the following terms have the meaning indicated in
order to facilitate the understanding of the present invention.
[0094] As used in the description and the appended claims, the
singular forms "a", "an", and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a composition" includes mixtures of two or more such
compositions, reference to "an inhibitor" includes mixtures of two
or more such inhibitors, and the like.
[0095] Unless stated otherwise, the term "about" means within 10%
(e.g., within 5%, 2% or 1%) of the particular value modified by the
term "about."
[0096] The transitional term "comprising," which is synonymous with
"including," "containing," or "characterized by," is inclusive or
open-ended and does not exclude additional, unrecited elements or
method steps. By contrast, the transitional phrase "consisting of"
excludes any element, step, or ingredient not specified in the
claim. The transitional phrase "consisting essentially of" limits
the scope of a claim to the specified materials or steps "and those
that do not materially affect the basic and novel
characteristic(s)" of the claimed invention.
[0097] With respect to compounds of the present invention, and to
the extent the following terms are used herein to further describe
them, the following definitions apply.
[0098] As used herein, the term "alkyl" refers to a saturated
linear or branched-chain monovalent hydrocarbon radical. In one
embodiment, the alkyl radical is a C1-C6 group. In other
embodiments, the alkyl radical is a C0-C6, C1-C6, C1-C5, C1-C4 or
C1-C3 group (wherein C0 alkyl refers to a bond). Examples of alkyl
groups include methyl, ethyl, 1-propyl, 2-propyl, i-propyl,
1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl,
n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl,
3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl,
2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl,
3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl,
3,3-dimethyl-2-butyl. In some embodiments, an alkyl group is a
C1-C3 alkyl group. In some embodiments, an alkyl group is a C1-C2
alkyl group.
[0099] As used herein, the term "alkylene" refers to a straight or
branched divalent hydrocarbon chain linking the rest of the
molecule to a radical group, consisting solely of carbon and
hydrogen, containing no unsaturation and having from one to 8
carbon atoms, for example, methylene, ethylene, propylene,
n-butylene, and the like. The alkylene chain may be attached to the
rest of the molecule through a single bond and to the radical group
through a single bond. In some embodiments, the alkylene group
contains one to 6 carbon atoms (C1-C6 alkylene). In other
embodiments, an alkylene group contains one to 5 carbon atoms
(C1-C5 alkylene). In other embodiments, an alkylene group contains
one to 4 carbon atoms (C1-C4 alkylene). In other embodiments, an
alkylene contains one to three carbon atoms (C1-C3 alkylene). In
other embodiments, an alkylene group contains one to two carbon
atoms (C1-C2 alkylene). In other embodiments, an alkylene group
contains one carbon atom (C1 alkylene).
[0100] As used herein, the term "ester" is represented by the
formula --OC(O)Z1 or --C(O)OZ1, where Z1 may be an alkyl,
halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl,
cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group, all as
described herein.
[0101] As used herein, the term "ether" is represented by the
formula Z1OZ2, where Z1 and Z2 can be, independently, an alkyl,
halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl,
cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group, all as
described herein.
[0102] As used herein, the term "carbocyclic" (also "carbocyclyl")
refers to a group that used alone or as part of a larger moiety,
contains a saturated, partially unsaturated, or aromatic ring
system having 5 to 6 carbon atoms, that is alone or part of a
larger moiety (e.g., an alkcarbocyclic group). In one embodiment,
carbocyclyl includes 5 to 6 carbon atoms (C5-C6). Representative
examples of monocyclic carbocyclyls include cyclopentyl and
cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl,
1-cyclohex-3-enyl, cyclohexadienyl and phenyl. The term carbocyclyl
includes aryl ring systems as defined herein. The term carbocycyl
also includes cycloalkyl rings (e.g., saturated or partially
unsaturated mono-, bi-, or spiro-carbocycles). The term carbocyclic
group also includes a carbocyclic ring fused to one or more (e.g.,
1, 2 or 3) different cyclic groups (e.g., aryl or heterocyclic
rings), where the radical or point of attachment is on the
carbocyclic ring.
[0103] Thus, the term carbocyclic also embraces carbocyclylalkyl
groups which as used herein refer to a group of the formula
-Rc-carbocyclyl where Rc is an alkylene chain. The term carbocyclic
also embraces carbocyclylalkoxy groups which as used herein refer
to a group bonded through an oxygen atom of the formula
--O-Rc-carbocyclyl where Rc is an alkylene chain.
[0104] As used herein, the term "heterocyclyl" refers to a
"carbocyclyl" that used alone or as part of a larger moiety,
contains a saturated, partially unsaturated or aromatic ring
system, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms have
been replaced with a heteroatom (e.g., O, N, N(O), S, S(O), or
S(O)2). The term heterocyclyl includes mono-, bi-, tri-, fused,
bridged, and spiro-ring systems, and combinations thereof. In some
embodiments, a heterocyclyl refers to a 5 to 6 membered
heterocyclyl ring system. In some embodiments, a heterocyclyl
refers to a saturated ring system, such as a 5 to 6 membered
saturated heterocyclyl ring system. In some embodiments, a
heterocyclyl refers to a heteroaryl ring system, such as a 5 to 6
membered heteroaryl ring system. The term heterocyclyl also
includes C5-C6 heterocycloalkyl, which is a saturated or partially
unsaturated ring system containing one or more heteroatoms.
[0105] In some embodiments, heterocyclyl includes 5-6 membered
monocycles. In some embodiments, the heterocyclyl group includes 0
to 3 double bonds. Examples of 5-membered heterocyclyls containing
a sulfur or oxygen atom and one to three nitrogen atoms are
thiazolyl, including thiazol-2-yl and thiazol-2-yl N-oxide,
thiadiazolyl, including 1,3,4-thiadiazol-5-yl and
1,2,4-thiadiazol-5-yl, oxazolyl, for example oxazol-2-yl, and
oxadiazolyl, such as 1,3,4-oxadiazol-5-yl, and
1,2,4-oxadiazol-5-yl. Example 5-membered ring heterocyclyls
containing 2 to 4 nitrogen atoms include imidazolyl, such as
imidazol-2-yl; triazolyl, such as 1,3,4-triazol-5-yl;
1,2,3-triazol-5-yl, 1,2,4-triazol-5-yl, and tetrazolyl, such as
1H-tetrazol-5-yl. Representative examples of benzo-fused 5-membered
heterocyclyls are benzoxazol-2-yl, benzthiazol-2-yl and
benzimidazol-2-yl. Example 6-membered heterocyclyls contain one to
three nitrogen atoms and optionally a sulfur or oxygen atom, for
example pyridyl, such as pyrid-2-yl, pyrid-3-yl, and pyrid-4-yl;
pyrimidyl, such as pyrimid-2-yl and pyrimid-4-yl; triazinyl, such
as 1,3,4-triazin-2-yl and 1,3,5-triazin-4-yl; pyridazinyl, in
particular pyridazin-3-yl, and pyrazinyl. The pyridine N-oxides and
pyridazine N-oxides and the pyridyl, pyrimid-2-yl, pyrimid-4-yl,
pyridazinyl and the 1,3,4-triazin-2-yl groups, are yet other
examples of heterocyclyl groups.
[0106] The term "selective degradation" refers to
ubiquitin-mediated degradation of a target protein, where the
target protein is degraded to a higher level relative to the at
least one other protein to which L.sub.1 binds. In some
embodiments, the heterobifunctional compound achieves degradation
of the target protein with substantially no degradation of the at
least one other protein.
[0107] The term "binding" as it relates to interaction between the
targeting ligand and the target protein, typically refers to an
inter-molecular interaction that is substantially specific in that
binding of the targeting ligand with other proteins that lack high
sequence identity to (e.g., are non-homologous with) the target
protein present in the cell is functionally insignificant. The term
"high sequence identity" as used herein refers to proteins that
share at least about 30%, about 35%, about 40%, about 50%, about
60%, about 70%, about 80%, about 90%, about 95%, or up to less than
100% sequence identity with the target protein. The term
"homologous" as used herein refers to a plurality of proteins
having a common lineage and which share, at least in a targeting
ligand binding portion thereof, a high sequence identity.
[0108] The term "binding" as it relates to interaction between the
degron and the E3 ubiquitin ligase, typically refers to an
inter-molecular interaction that may or may not exhibit an affinity
level that equals or exceeds that affinity between the targeting
ligand and the target protein, but nonetheless wherein the affinity
is sufficient to achieve recruitment of the ligase to the targeted
degradation and the selective degradation of the targeted
protein.
[0109] The term "binding conformation" refers to the spatial
relationship between proteins that are bound to each other, and may
be represented in terms of shortest path distance between the bound
proteins, orientation of one protein with respect to the other
protein, inter-molecular interactions (e.g., binding affinity or
energy level) between the proteins, identification of amino acid
residues that form the inter-molecular bonds between the two
proteins, and/or any other information that can represent the
spatial relationship between the two proteins in empirical
terms.
[0110] The term "ligand-induced dimerization" refers to bringing
together of two proteins within close proximity to one-another by
means of binding each of the two proteins to a ligand, where the
two ligands may be part of the same compound (e.g.,
heterobifunctional compound). The proximity between the two
proteins may be sufficient to enable one of the two proteins to
functionally act on the other protein (e.g., one protein
enzymatically modifying the other protein or degrading the other
protein).
[0111] The term "homologous proteins" refers to a plurality of
proteins that due to their common lineage, share similar amino acid
sequences, of at least portions (e.g., functional domains or
epitopes) of proteins, where the extent of similarity between the
amino acid sequences of the proteins is significantly higher than
that the similarity that is expected from two completely unrelated
proteins.
[0112] The heterobifunctional compounds of the present invention
are represented by Formula (I):
##STR00002##
wherein n is 0 or 1; m is 0 or 1; p is 0 or 1; and R.sub.1 is an
ether, an alkyl ether, an alkyl amine, C.sub.1 alkyl, C.sub.2
alkyl, C.sub.3 alkyl, C.sub.4 alkyl, C.sub.5 alkyl, C.sub.6 alkyl,
or a 5- or 6-member cyclic group; wherein L.sub.1 binds a target
protein and at least one other protein; and wherein L.sub.2 binds
an E3 ubiquitin ligase or a component of an E3 ubiquitin ligase, or
a pharmaceutically acceptable salt, ester or stereoisomer thereof.
In some embodiments, the at least one other protein to which
L.sub.1 binds has a high sequence identity with the target protein.
In some embodiments, the at least one other protein to which
L.sub.1 binds is homologous to the target protein.
[0113] The linker has a structure represented by formula (II):
##STR00003##
wherein n is 0 or 1; m is 0 or 1; p is 0 or 1; and R.sub.1 is an
ether (e.g., a polyethylene glycol chain ranging from 1 to 2
ethylene glycol units), an alkyl ether, an alkyl amine, C1-6
alkylene or a 5- to 6-membered carbocyclic or heterocyclic
group.
[0114] In various embodiments, the linker of formula (II) is
represented by any of the following structures:
##STR00004##
[0115] In various embodiments, the target protein is a member of
BET family bromodomain-containing proteins. In various embodiments,
the target protein is BRD4. A representative example of a targeting
ligand (L.sub.1) that binds BRD4 is JQ1 (Structure 1) or an analog
thereof.
##STR00005##
[0116] Representative examples of L.sub.1 which are analogs of JQ1
have the following structures:
##STR00006##
[0117] In various embodiments, L.sub.1 is a thiophene analog of
JQ1. represented by structure 2:
##STR00007##
wherein
##STR00008##
wherein R.sub.3 is methyl or
##STR00009##
R.sub.2 is
##STR00010##
[0118] and R.sub.4 is
##STR00011##
[0120] Accordingly, in various embodiments the compound of formula
(I) includes a ligand L.sub.1 that is represented by any of the
following structures:
##STR00012##
[0121] In various embodiments, L.sub.2 is an IMiD (e.g.,
thalidomide, lenalidomide or pomalidomide or an analog
thereof).
[0122] In various embodiments, L.sub.2 is represented by any of the
following structures:
##STR00013## ##STR00014##
[0123] Thus, in some embodiments, the compounds of the present
invention are represented by any structures of formula I, wherein
L.sub.1 is represented by any structures L.sub.1 described herein,
structure 1, structure 1-a to Structure 1-c and structure 2, the
linker is represented by any of the structures C1-a to C1-J, and
L.sub.2 is represented by any of the structures L2-a to L2-I, or a
pharmaceutically acceptable salt or stereoisomer thereof.
[0124] In various embodiments, the bifunctional compound of formula
(I) is represented by any of the following structures:
##STR00015## ##STR00016## ##STR00017## ##STR00018##
and pharmaceutically acceptable salts, esters and stereoisomers
thereof.
[0125] Compounds of the present invention (which as used
hereinafter, refer to both immunomodulatory compounds of formula
(I) and the degraders of formula (I)) may be in the form of a free
acid or free base, or a pharmaceutically acceptable salt or ester.
As used herein, the term "pharmaceutically acceptable" in the
context of a salt or ester refers to a salt or ester of the
compound that does not abrogate the biological activity or
properties of the compound, and is relatively non-toxic, i.e., the
compound in salt form may be administered to a subject without
causing undesirable biological effects (such as dizziness or
gastric upset) or interacting in a deleterious manner with any of
the other components of the composition in which it is contained.
The term "pharmaceutically acceptable salt or ester" refers to a
product obtained by reaction of the compound of the present
invention with a suitable acid or a base. Examples of
pharmaceutically acceptable salts of the compounds of this
invention include those derived from suitable inorganic bases such
as Li, Na, K, Ca, Mg, Fe, Cu, Al, Zn and Mn salts. Examples of
pharmaceutically acceptable, nontoxic acid addition salts are salts
of an amino group formed with inorganic acids such as
hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate,
bisulfate, phosphate, isonicotinate, acetate, lactate, salicylate,
citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate,
maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate,
formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,
benzenesulfonate, 4-methylbenzenesulfonate or p-toluenesulfonate
salts and the like. Certain compounds of the invention can form
pharmaceutically acceptable salts with various organic bases such
as lysine, arginine, guanidine, diethanolamine or metformin.
Suitable base salts include aluminum, calcium, lithium, magnesium,
potassium, sodium, or zinc, salts. Representative examples of
pharmaceutically acceptable esters include (e.g., methyl, ethyl,
isopropyl and tert-butyl esters).
[0126] In some embodiments, the compounds of the present invention
is an isotopic derivative in that it has at least one desired
isotopic substitution of an atom, at an amount above the natural
abundance of the isotope, i.e., enriched. In one embodiment, the
compound includes deuterium or multiple deuterium atoms.
Substitution with heavier isotopes such as deuterium, i.e. .sup.2H,
may afford certain therapeutic advantages resulting from greater
metabolic stability, for example, increased in vivo half-life or
reduced dosage requirements, and thus may be advantageous in some
circumstances. For example, in compounds of formula (I) that target
BRD4, a JQ1 moiety may be deuterated in order to increase
half-life.
[0127] Compounds of the present invention may have at least one
chiral center and thus may be in the form of a stereoisomer, which
as used herein, embraces all isomers of individual compounds that
differ only in the orientation of their atoms in space. The term
stereoisomer includes mirror image isomers (enantiomers which
include the (R-) or (S-) configurations of the compounds), mixtures
of mirror image isomers (physical mixtures of the enantiomers, and
racemates or racemic mixtures) of compounds, geometric (cis/trans
or E/Z, R/S) isomers of compounds and isomers of compounds with
more than one chiral center that are not mirror images of one
another (diastereoisomers). The chiral centers of the compounds may
undergo epimerization in vivo; thus, for these compounds,
administration of the compound in its (R-) form is considered
equivalent to administration of the compound in its (S-) form.
Accordingly, the compounds of the present invention may be made and
used in the form of individual isomers and substantially free of
other isomers, or in the form of a mixture of various isomers,
e.g., racemic mixtures of stereoisomers.
[0128] In addition, the compounds of the present invention embrace
the use of N-oxides, crystalline forms (also known as polymorphs),
active metabolites of the compounds having the same type of
activity, tautomers, and unsolvated as well as solvated forms with
pharmaceutically acceptable solvents such as water, ethanol, and
the like, of the compounds. The solvated forms of the conjugates
presented herein are also considered to be disclosed herein.
[0129] Without intending to be bound by any particular theory of
operation, it is believed that the heterobifunctional compound
mediates the ligand-induced dimerization of the target protein and
the E3 ubiquitin ligase or the component of the E3 ubiquitin
ligase, such that the binding conformation between the target
protein and the E3 ubiquitin ligase or the component of the E3
ubiquitin ligase result distinct mutational signature of binding,
and that, the binding affinity of the heterobifunctional compound
to the E3 ubiquitin ligase or the component of E3 ubiquitin ligase
is reduced when the heterobifunctional compound is bound to the
target protein (e.g., the DC.sub.50/5h is about 500 nM or less,
about 50 nM or less, about 10 nM or less, about 5 nM or less, or
about 1 nM or less).
[0130] Methods of Synthesis
[0131] Broadly, the inventive compounds or
pharmaceutically-acceptable salts, esters or stereoisomers thereof,
may be prepared by any process known to be applicable to the
preparation of chemically related compounds. The compounds of the
present invention will be better understood in connection with the
synthetic schemes that are described in various working examples
and which illustrate non-limiting methods by which the compounds of
the invention may be prepared.
[0132] Pharmaceutical Compositions
[0133] The compounds of the present invention may be formulated
into several different types of pharmaceutical compositions that
contain a therapeutically effective amount of the compound, and a
pharmaceutically acceptable carrier. Generally, the inventive
compounds may be formulated into a given type of composition in
accordance with conventional pharmaceutical practice such as
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping and compression
processes (see, e.g., Remington: The Science and Practice of
Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams &
Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds.
J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New
York).
[0134] The term "pharmaceutically acceptable carrier," as known in
the art, refers to a pharmaceutically acceptable material,
composition or vehicle, suitable for administering compounds of the
present invention to mammals. Suitable carriers may include, for
example, liquids (both aqueous and non-aqueous alike, and
combinations thereof), solids, encapsulating materials, gases, and
combinations thereof (e.g., semi-solids), and gases, that function
to carry or transport the compound from one organ, or portion of
the body, to another organ, or portion of the body. A carrier is
"acceptable" in the sense of being physiologically inert to and
compatible with the other ingredients of the formulation and not
injurious to the subject or patient. Depending on the type of
formulation, the composition may include one or more
pharmaceutically acceptable excipients.
[0135] Accordingly, compounds of the present invention may be
formulated into solid compositions (e.g., powders, tablets,
dispersible granules, capsules, cachets, and suppositories), liquid
compositions (e.g., solutions in which the compound is dissolved,
suspensions in which solid particles of the compound are dispersed,
emulsions, and solutions containing liposomes, micelles, or
nanoparticles, syrups and elixirs); semi-solid compositions (e.g.,
gels, suspensions and creams); and gases (e.g., propellants for
aerosol compositions). Compounds may also be formulated for rapid,
intermediate or extended release.
[0136] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active compound is mixed with a carrier such as sodium citrate
or dicalcium phosphate and an additional carrier or excipient such
as a) fillers or extenders such as starches, lactose, sucrose,
glucose, mannitol, and silicic acid, b) binders such as, for
example, methylcellulose, microcrystalline cellulose,
hydroxypropylmethylcellulose, carboxymethylcellulose, sodium
carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,
sucrose, and acacia, c) humectants such as glycerol, d)
disintegrating agents such as crosslinked polymers (e.g.,
crosslinked polyvinylpyrrolidone (crospovidone), crosslinked sodium
carboxymethyl cellulose (croscarmellose sodium), sodium starch
glycolate, agar-agar, calcium carbonate, potato or tapioca starch,
alginic acid, certain silicates, and sodium carbonate, e) solution
retarding agents such as paraffin, f) absorption accelerators such
as quaternary ammonium compounds, g) wetting agents such as, for
example, cetyl alcohol and glycerol monostearate, h) absorbents
such as kaolin and bentonite clay, and i) lubricants such as talc,
calcium stearate, magnesium stearate, solid polyethylene glycols,
sodium lauryl sulfate, and mixtures thereof. In the case of
capsules, tablets and pills, the dosage form may also include
buffering agents. Solid compositions of a similar type may also be
employed as fillers in soft and hard-filled gelatin capsules using
such excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings and other
coatings. They may further contain an opacifying agent.
[0137] In some embodiments, compounds of the present invention may
be formulated in a hard or soft gelatin capsule. Representative
excipients that may be used include pregelatinized starch,
magnesium stearate, mannitol, sodium stearyl fumarate, lactose
anhydrous, microcrystalline cellulose and croscarmellose sodium.
Gelatin shells may include gelatin, titanium dioxide, iron oxides
and colorants.
[0138] To the extent that compounds of the present invention are
water-soluble, they may be formulated as solutions for parenteral
and oral delivery forms. Parenteral administration may also be
advantageous in that the compound may be administered relatively
quickly such as in the case of a single-dose treatment and/or an
acute condition.
[0139] Injectable preparations for parenteral administration may
include sterile aqueous solutions or oleaginous suspensions. They
may be formulated according to standard techniques using suitable
dispersing or wetting agents and suspending agents. The sterile
injectable preparation may also be a sterile injectable solution,
suspension or emulsion in a nontoxic parenterally acceptable
diluent or solvent, for example, as a solution in 1,3-butanediol.
Among the acceptable vehicles and solvents that may be employed are
water, Ringer's solution, U.S.P. and isotonic sodium chloride
solution. In addition, sterile, fixed oils are conventionally
employed as a solvent or suspending medium. For this purpose any
bland fixed oil can be employed including synthetic mono- or
diglycerides. In addition, fatty acids such as oleic acid are used
in the preparation of injectables. The injectable formulations can
be sterilized, for example, by filtration through a
bacterial-retaining filter, or by incorporating sterilizing agents
in the form of sterile solid compositions which can be dissolved or
dispersed in sterile water or other sterile injectable medium prior
to use. The effect of the compound may be prolonged by slowing its
absorption, which may be accomplished by the use of a liquid
suspension or crystalline or amorphous material with poor water
solubility. Prolonged absorption of the compound from a
parenterally administered formulation may also be accomplished by
suspending the compound in an oily vehicle.
[0140] In certain embodiments, compounds of the present invention
may be administered in a local rather than systemic manner, for
example, via injection of the conjugate directly into an organ,
often in a depot preparation or sustained release formulation. In
specific embodiments, long acting formulations are administered by
implantation (for example subcutaneously or intramuscularly) or by
intramuscular injection. Injectable depot forms are made by forming
microencapsule matrices of the compound in a biodegradable polymer,
e.g., polylactide-polyglycolides, poly(orthoesters) and
poly(anhydrides). The rate of release of the compound may be
controlled by varying the ratio of compound to polymer and the
nature of the particular polymer employed. Depot injectable
formulations are also prepared by entrapping the compound in
liposomes or microemulsions that are compatible with body tissues.
Furthermore, in other embodiments, the compound is delivered in a
targeted drug delivery system, for example, in a liposome coated
with organ-specific antibody. In such embodiments, the liposomes
are targeted to and taken up selectively by the organ.
[0141] Liquid dosage forms for oral administration include
solutions, suspensions, emulsions, micro-emulsions, syrups and
elixirs. In addition to the compound, the liquid dosage forms may
contain an aqueous or non-aqueous carrier (depending upon the
solubility of the compounds) commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Oral compositions may also include an excipients such as wetting
agents, suspending agents, coloring, sweetening, flavoring, and
perfuming agents.
[0142] Other routes of administration that may be suitable for the
compounds of the present invention include buccal, inhalation,
topical, transdermal, transmucosal, ophthalmic, rectal and
vaginal.
[0143] The compositions may be formulated for buccal or sublingual
administration, examples of which include tablets, lozenges and
gels.
[0144] The compositions may be formulated for administration by
inhalation. Various forms suitable for administration by inhalation
include aerosols, mists or powders. Pharmaceutical compositions may
be delivered in the form of an aerosol spray presentation from
pressurized packs or a nebulizer, with the use of a suitable
propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
In some embodiments, the dosage unit of a pressurized aerosol may
be determined by providing a valve to deliver a metered amount. In
some embodiments, capsules and cartridges including gelatin, for
example, for use in an inhaler or insufflator, may be formulated
containing a powder mix of the compound and a suitable powder base
such as lactose or starch.
[0145] Compounds may be formulated for topical administration which
as used herein, refers to administration intradermally by
application of the formulation to the epidermis. These types of
compositions are typically in the form of ointments, pastes,
creams, lotions, gels, solutions and sprays.
[0146] Representative examples of carriers useful in formulating
compositions for topical application include solvents (e.g.,
alcohols, poly alcohols, water), creams, lotions, ointments, oils,
plasters, liposomes, powders, emulsions, microemulsions, and
buffered solutions (e.g., hypotonic or buffered saline). Creams,
for example, may be formulated using saturated or unsaturated fatty
acids such as stearic acid, palmitic acid, oleic acid,
palmito-oleic acid, and cetyl or oleyl alcohols. Creams may also
contain a non-ionic surfactant such as polyoxy-40-stearate.
[0147] In some embodiments, the topical formulations may also
include an excipient, an example of which is a penetration
enhancing agent. These agents are capable of transporting a
pharmacologically active compound through the stratum corneum and
into the epidermis or dermis, preferably, with little or no
systemic absorption. A wide variety of compounds have been
evaluated as to their effectiveness in enhancing the rate of
penetration of drugs through the skin. See, for example,
Percutaneous Penetration Enhancers, Maibach H. I. and Smith H. E.
(eds.), CRC Press, Inc., Boca Raton, Fla. (1995), which surveys the
use and testing of various skin penetration enhancers, and
Buyuktimkin et al., Chemical Means of Transdermal Drug Permeation
Enhancement in Transdermal and Topical Drug Delivery Systems, Gosh
T. K., Pfister W. R., Yum S. I. (Eds.), Interpharm Press Inc.,
Buffalo Grove, Ill. (1997). Representative examples of penetration
enhancing agents include triglycerides (e.g., soybean oil), aloe
compositions (e.g., aloe-vera gel), ethyl alcohol, isopropyl
alcohol, octolyphenylpolyethylene glycol, oleic acid, polyethylene
glycol 400, propylene glycol, N-decylmethylsulfoxide, fatty acid
esters (e.g., isopropyl myristate, methyl laurate, glycerol
monooleate, and propylene glycol monooleate), and
N-methylpyrrolidone.
[0148] Representative examples of yet other excipients that may be
included in topical as well as in other types of formulations (to
the extent they are compatible), include preservatives,
antioxidants, moisturizers, emollients, buffering agents,
solubilizing agents, skin protectants, and surfactants. Suitable
preservatives include alcohols, quaternary amines, organic acids,
parabens, and phenols. Suitable antioxidants include ascorbic acid
and its esters, sodium bisulfite, butylated hydroxytoluene,
butylated hydroxyanisole, tocopherols, and chelating agents like
EDTA and citric acid. Suitable moisturizers include glycerine,
sorbitol, polyethylene glycols, urea, and propylene glycol.
Suitable buffering agents include citric, hydrochloric, and lactic
acid buffers. Suitable solubilizing agents include quaternary
ammonium chlorides, cyclodextrins, benzyl benzoate, lecithin, and
polysorbates. Suitable skin protectants include vitamin E oil,
allatoin, dimethicone, glycerin, petrolatum, and zinc oxide.
[0149] Transdermal formulations typically employ transdermal
delivery devices and transdermal delivery patches wherein the
compound is formulated in lipophilic emulsions or buffered, aqueous
solutions, dissolved and/or dispersed in a polymer or an adhesive.
Patches may be constructed for continuous, pulsatile, or on demand
delivery of pharmaceutical agents. Transdermal delivery of the
compounds may be accomplished by means of an iontophoretic patch.
Transdermal patches may provide controlled delivery of the
compounds wherein the rate of absorption is slowed by using
rate-controlling membranes or by trapping the compound within a
polymer matrix or gel. Absorption enhancers may be used to increase
absorption, examples of which include absorbable pharmaceutically
acceptable solvents that assist passage through the skin.
[0150] Ophthalmic formulations include eye drops.
[0151] Formulations for rectal administration include enemas,
rectal gels, rectal foams, rectal aerosols, and retention enemas,
which may contain conventional suppository bases such as cocoa
butter or other glycerides, as well as synthetic polymers such as
polyvinylpyrrolidone, PEG, and the like. Compositions for rectal or
vaginal administration may also be formulated as suppositories
which can be prepared by mixing the compound with suitable
non-irritating carriers and excipients such as cocoa butter,
mixtures of fatty acid glycerides, polyethylene glycol, suppository
waxes, and combinations thereof, all of which are solid at ambient
temperature but liquid at body temperature and therefore melt in
the rectum or vaginal cavity and release the compound.
[0152] Dosage Amounts
[0153] As used herein, the term, "therapeutically effective amount"
refers to an amount of a compound of the application or a
pharmaceutically acceptable salt or a stereoisomer thereof; or a
composition including the compound of the application or a
pharmaceutically acceptable salt or a stereoisomer thereof,
effective in producing the desired therapeutic response in a
particular patient suffering from a disease or disorder mediated by
the dysfunctional or dysregulated target protein. The term
"therapeutically effective amount" includes the amount of the
compound of the application or a pharmaceutically acceptable salt
or a stereoisomer thereof, when administered, may induce a positive
modification in the disease or disorder to be treated (e.g.,
remission), or is sufficient to prevent development or progression
of the disease or disorder, or alleviate to some extent, one or
more of the symptoms of the disease or disorder being treated in a
subject. In respect of the therapeutic amount of the compound, the
amount of the compound used for the treatment of a subject is low
enough to avoid undue or severe side effects, within the scope of
sound medical Judgment can also be considered. The therapeutically
effective amount of the compound or composition will be varied with
the particular condition being treated, the severity of the
condition being treated or prevented, the duration of the
treatment, the nature of concurrent therapy, the age and physical
condition of the end user, the specific compound or composition
employed and the particular pharmaceutically acceptable carrier
utilized.
[0154] The total daily dosage of the compounds and usage thereof
may be decided in accordance with standard medical practice, e.g.,
by the attending physician using sound medical judgment. The
specific therapeutically effective dose for any particular patient
will depend upon a variety of factors including the disease or
disorder being treated and the severity thereof (e.g., its present
status); the activity of the specific compound employed; the
specific composition employed; the age, body weight, general
health, sex and diet of the patient; the time of administration,
route of administration, and rate of excretion of the specific
compound employed; the duration of the treatment; drugs used in
combination or coincidental with the specific compound employed;
and like factors well known in the medical arts (see, for example,
Goodman and Gilman's, "The Pharmacological Basis of Therapeutics",
10th Edition, A. Gilman, J. Hardman and L. Limbird, eds.,
McGraw-Hill Press, 155-173, 2001).
[0155] In some embodiments, the therapeutic regimens include
titrating the dosages administered to the patient so as to achieve
a specified measure of therapeutic efficacy. Such measures include
a reduction in the cancer cell population in the patient. In
treating certain human patients having solid tumors, for example,
extracting multiple tissue specimens from a suspected tumor site
may prove impracticable. In these cases, the dosage of the compound
for a human patient may be extrapolated from doses in animal models
that are effective to reduce the cancer population in those animal
models. In the animal models, the treatment may be adjusted so as
to achieve a reduction in the number or amount of cancer cells
found in a test specimen extracted from an animal after undergoing
the treatment, as compared with a reference sample. The reference
sample can be a specimen extracted from the same animal, prior to
receiving the treatment. In specific embodiments, the number or
amount of cancer cells in the test specimen is at least 2%, 5%,
10%, 15%, 20%, 30%, 40%, 50% or 60% lower than in the reference
sample. The doses effective in reducing the number or amount of
cancer cells in the animals can be normalized to body surface area
(e.g., mg/m.sup.2) to provide an equivalent human dose.
[0156] Compounds of the present invention may be effective over a
wide dosage range. In some embodiments, the total daily dosage
(e.g., for adult humans) may range from about 0.001 to about 1000
mg, from 0.01 to about 1000 mg, from 0.01 to about 500 mg, from
about 0.01 to about 100 mg, from about 0.5 to about 100 mg, from 1
to about 100-400 mg per day, from about 1 to about 50 mg per day,
and from about 5 to about 40 mg per day, and in yet other
embodiments from about 10 to about 30 mg per day. Individual dosage
may be formulated to contain the desired dosage amount depending
upon the number of times the compound is administered per day. By
way of example, capsules may be formulated with from about 1 to
about 200 mg of compound (e.g., 1, 2, 2.5, 3, 4, 5, 10, 15, 20, 25,
50, 100, 150, and 200 mgs).
[0157] Methods of Use
[0158] The compounds of the present invention may be useful in the
treatment of diseases and disorders wherein a dysfunctional or
dysregulated protein (that can be targeted for degradation by
cereblon, participates in the inception, manifestation of one or
more symptoms or markers, severity or progression of the disease or
disorder), and where the degradation of the targeted protein may
confer a therapeutic benefit. The diseases or disorders may be said
to be characterized or mediated by dysfunctional or dysregulated
protein activity (e.g., elevated protein levels compared to a
non-pathological state). A "disease" is generally regarded as a
state of health of an animal wherein the animal cannot maintain
homeostasis, and wherein if the disease is not ameliorated then the
animal's health continues to deteriorate. In contrast, a "disorder"
in an animal is a state of health in which the animal is able to
maintain homeostasis, but in which the animal's state of health is
less favorable than it would be in the absence of the disorder.
Left untreated, a disorder does not necessarily cause a further
decrease in the animal's state of health. In some embodiments,
compounds of the application may be useful in the treatment of
proliferative diseases and disorders (e.g., cancer or benign
neoplasms). As used herein, the term "cell proliferative disease or
disorder" refers to the conditions characterized by unregulated or
abnormal cell growth, or both. Cell proliferative disorders include
noncancerous conditions, precancerous conditions, and cancer.
[0159] The present methods thus include administering a
therapeutically effective amount of a compound to a subject in need
thereof. The term "subject" as used herein includes all members of
the animal kingdom prone to or suffering from the indicated disease
or disorder. In some embodiments, the subject is a mammal, e.g., a
human or a non-human mammal. The methods are also applicable to
companion animals such as dogs and cats as well as livestock such
as cows, horses, sheep, goats, pigs, and other domesticated and
wild animals. A subject "suffering from or suspected of suffering
from" a specific disease or disorder may have a sufficient number
of risk factors or presents with a sufficient number or combination
of signs or symptoms such that a medical professional could
diagnose or suspect that the subject was suffering from the disease
or disorder. Methods for identification of subjects suffering from
or suspected of suffering from conditions associated with cancer is
within the ability of those in the art. Subjects suffering from,
and suspected of suffering from, a specific disease, condition, or
syndrome are not necessarily two distinct groups. For purposes of
the present application, "subjects" and "patients" are used
interchangeably.
[0160] In general, methods of using the compounds of the present
invention include administering to a subject in need thereof a
therapeutically effective amount of a compound of the present
invention.
[0161] Exemplary types of non-cancerous diseases or disorders that
may be amenable to treatment with the compounds of the present
invention include inflammatory diseases and conditions, autoimmune
diseases, heart diseases, viral diseases, chronic and acute kidney
diseases or injuries, obesity, metabolic diseases, allergic and
genetic diseases.
[0162] Representative examples of specific non-cancerous diseases
and disorders include rheumatoid arthritis, inflammation,
lymphoproliferative conditions, acromegaly, rheumatoid spondylitis,
osteoarthritis, gout, sepsis, septic shock, endotoxic shock,
gram-negative sepsis, toxic shock syndrome, asthma, adult
respiratory distress syndrome, chronic obstructive pulmonary
disease, chronic pulmonary inflammation, inflammatory bowel
disease, Crohn's disease, systemic lupus erythematosus, multiple
sclerosis, juvenile-onset diabetes, systemic lupus erythematosus,
autoimmune uveoretinitis, autoimmune vasculitis, bullous pemphigus,
myasthenia gravis, autoimmune thyroditis or Hashimoto's disease,
Sjogren's syndrome, granulomatous orchitis, autoimmune oophoritis,
sarcoidosis, rheumatic carditis, ankylosing spondylitis, Grave's
disease, autoimmune thrombocytopenic purpura, psoriasis, eczema,
ulcerative colitis, pancreatic fibrosis, hepatic fibrosis, acute
and chronic renal disease, irritable bowel syndrome, pyresis,
restenosis, cerebral malaria, stroke and ischemic injury, neural
trauma, Alzheimer's disease, Huntington's disease, Parkinson's
disease, acute and chronic pain, allergic rhinitis, allergic
conjunctivitis, chronic heart failure, congestive heart failure,
acute coronary syndrome, cachexia, malaria, leprosy, leishmaniasis,
Lyme disease, Reiter's syndrome, acute synovitis, muscle
degeneration, bursitis, tendonitis, tenosynovitis, herniated,
ruptures, or prolapsed intervertebral disk syndrome, osteopetrosis,
thrombosis, restenosis, silicosis, pulmonary sarcosis, bone
resorption diseases, such as osteoporosis, graft-versus-host
reaction, Multiple Sclerosis, lupus, fibromyalgia, AIDS and other
viral diseases such as Herpes Zoster, Herpes Simplex I or II,
influenza virus and cytomegalovirus, diabetes Type I and II,
obesity, insulin resistance and diabetic retinopathy, 22q11.2
deletion syndrome, Angelman syndrome, Canavan disease, celiac
disease, Charcot-Marie-Tooth disease, color blindness, Cri du chat,
down syndrome, cystic fibrosis, Duchenne muscular dystrophy,
haemophilia, Klinefelter's syndrome, neurofibromatosis,
phenylketonuria, Prader-Willi syndrome, sickle cell disease,
Tay-Sachs disease, Turner syndrome, urea cycle disorders,
thalassemia, cystic fibrosis, rheumatoid arthritis, Sjogren's
syndrome, uveitis, polymyositis, and dermatomyositis,
arteriosclerosis, amyotrophic lateral sclerosis, asociality,
affective disorders, systemic lupus erythematosus, immune response,
varicosis, vaginitis, including chronic recurrent yeast vaginitis,
depression, Sudden Infant Death Syndrome, and varicosis.
[0163] In other embodiments, the methods are directed to treating
subjects having cancer. Broadly, the compounds of the present
invention may be effective in the treatment of carcinomas (solid
tumors including both primary and metastatic tumors), sarcomas,
melanomas, and hematological cancers (cancers affecting blood
including lymphocytes, bone marrow and/or lymph nodes) including
leukemia, lymphoma and multiple myeloma. Adult tumors/cancers and
pediatric tumors/cancers are included. The cancers may be
vascularized, or not yet substantially vascularized, or
non-vascularized tumors.
[0164] Representative examples of cancers includes adrenocortical
carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal
cancer, anorectal cancer, cancer of the anal canal, appendix
cancer, childhood cerebellar astrocytoma, childhood cerebral
astrocytoma, basal cell carcinoma, skin cancer (non-melanoma),
biliary cancer, extrahepatic bile duct cancer, intrahepatic bile
duct cancer, bladder cancer, urinary bladder cancer, cone and joint
cancer, brain cancer (e.g., brain stem glioma, cerebellar
astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma,
medulloblastoma, supratentorial primitive neuroectodeimal tumors,
visual pathway and hypothalamic glioma), breast cancer, bronchial
adenomas/carcinoids, carcinoid tumor, gastrointestinal, nervous
system cancer, nervous system lymphoma, central nervous system
cancer, central nervous system lymphoma, cervical cancer, childhood
cancers, chronic lymphocytic leukemia, chronic myelogenous
leukemia, chronic myeloproliferative disorders, colon cancer,
rectal cancer, cutaneous T-cell lymphoma, lymphoid neoplasm,
mycosis fungoids, Sezary Syndrome, endometrial cancer, esophageal
cancer, extracranial germ cell tumor, extragonadal germ cell tumor,
extrahepatic bile duct cancer, eye cancer, intraocular melanoma,
retinoblastoma, gallbladder cancer, gastric (stomach) cancer,
gastrointestinal carcinoid tumor, gastrointestinal stromal tumor
(GIST), germ cell tumor, ovarian germ cell tumor, gestational
trophoblastic tumor glioma, head and neck cancer, hepatocellular
(liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer,
intraocular melanoma, ocular cancer, islet cell tumors (endocrine
pancreas), Kaposi Sarcoma, renal cancer, kidney cancer, clear cell
renal cell carcinoma, laryngeal cancer, acute lymphoblastic
leukemia, acute myeloid leukemia, chronic lymphocytic leukemia,
chronic myelogenous leukemia, hair cell leukemia, lip and oral
cavity cancer, liver cancer, lung cancer, non-small cell lung
cancer, small cell lung cancer, AIDS-related lymphoma,
non-Hodgkin's lymphoma, primary central nervous system lymphoma,
Waldenstrom's macroglobulinemia, medulloblastoma, melanoma,
intraocular (eye) melanoma, merkel cell carcinoma, mesothelioma
malignant, mesothelioma, metastatic squamous neck cancer, mouth
cancer, cancer of the tongue, multiple endocrine neoplasia
syndrome, mycosis fungoids, myelodysplastic syndromes,
myelodysplastic/myeloproliferative diseases, acute myeloid
leukemia, multiple myeloma, chromic myeloproliferative disorders,
nasopharyngeal cancer, neuroblastoma, oral cancer, oral cavity
cancer, oropharyngeal cancer, ovarian cancer (e.g., ovarian
epithelial cancer, ovarian low malignant potential tumor),
pancreatic cancer, islet cell pancreatic cancer, paranasal sinus
and nasal cavity cancer, parathyroid cancer, penile cancer,
pharyngeal cancer, pheochromocytoma, pineoblastoma and
supratentorial primitive neuroectodermal tumors, pituitary tumor,
plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma,
prostate cancer, rectal cancer, renal pelvis and ureter,
transitional cell cancer, retinoblastoma rhabdomyosarcoma, salivary
gland cancer, Ewing family of sarcoma tumors, Kaposi Sarcoma, soft
tissue sarcoma, uterine cancer, merkel cell skin carcinoma, small
intestine cancer, squamous cell carcinoma, stomach (gastric)
cancer, supratentorial primitive neuroectodermal tumors, testicular
cancer, throat cancer, thymoma, thymoma and thymic carcinoma,
thyroid cancer, transitional cell cancer of the renal pelvis and
ureter and other urinary organs, gestational trophoblastic tumor,
urethral cancer, endometrial uterine cancer, uterine sarcoma,
uterine corpus cancer, vaginal cancer, vulvar cancer, and Wilms'
Tumor.
[0165] Sarcomas that may be treatable with compounds of the present
invention include both soft tissue and bone cancers alike,
representative examples of which include osteosarcoma or osteogenic
sarcoma (bone), chondrosarcoma (cartilage), leiomyosarcoma (smooth
muscle), rhabdomyosarcoma (skeletal muscle), mesothelial sarcoma or
mesothelioma (membranous lining of body cavities), fibrosarcoma
(fibrous tissue), angiosarcoma or hemangioendothelioma (blood
vessels), liposarcoma (adipose tissue), glioma or astrocytoma
(neurogenic connective tissue found in the brain), myxosarcoma
(primitive embryonic connective tissue) and mesenchymous or mixed
mesodermal tumor (mixed connective tissue types).
[0166] In some embodiments, methods of the present invention entail
treatment of subjects having cell proliferative diseases or
disorders of the hematological system, liver (hepatocellular),
brain, lung, colorectal (e.g., colon), pancreas, prostate, skin,
ovary, breast, skin (e.g., melanoma), and endometrium.
[0167] As used herein, "cell proliferative diseases or disorders of
the hematologic system" include lymphoma, leukemia, myeloid
neoplasms, mast cell neoplasms, myelodysplasia, benign monoclonal
gammopathy, lymphomatoid papulosis, polycythemia vera, chronic
myelocytic leukemia, agnogenic myeloid metaplasia, and essential
thrombocythemia. Representative examples of hematologic cancers may
thus include multiple myeloma, lymphoma (including T-cell lymphoma,
Hodgkin's lymphoma, non-Hodgkin's lymphoma (diffuse large B-cell
lymphoma (DLBCL), follicular lymphoma (FL), acute myeloid leukemia
(AML), mantle cell lymphoma (MCL) and ALK+ anaplastic large cell
lymphoma) (e.g., B-cell non-Hodgkin's lymphoma selected from
diffuse large B-cell lymphoma (e.g., germinal center B-cell-like
diffuse large B-cell lymphoma or activated B-cell-like diffuse
large B-cell lymphoma), Burkitt's lymphoma/leukemia, mantle cell
lymphoma, mediastinal (thymic) large B-cell lymphoma, follicular
lymphoma, marginal zone lymphoma, lymphoplasmacytic
lymphoma/Waldenstrom macroglobulinemia, refractory B-cell
non-Hodgkin's lymphoma, and relapsed B-cell non-Hodgkin's
lymphoma), childhood lymphomas, and lymphomas of lymphocytic and
cutaneous origin, e.g., small lymphocytic lymphoma), leukemia
(including childhood leukemia, hairy-cell leukemia, acute
lymphocytic leukemia, acute myelocytic leukemia, acute myeloid
leukemia (e.g., acute monocytic leukemia), chronic lymphocytic
leukemia, small lymphocytic leukemia, chronic myelocytic leukemia,
chronic myelogenous leukemia, and mast cell leukemia), myeloid
neoplasms and mast cell neoplasms.
[0168] As used herein, "cell proliferative diseases or disorders of
the lung" include all forms of cell proliferative disorders
affecting lung cells. Cell proliferative disorders of the lung
include lung cancer, a precancer or precancerous condition of the
lung, benign growths or lesions of the lung, and metastatic lesions
in the tissue and organs in the body other than the lung. Lung
cancer includes all forms of cancer of the lung, e.g., malignant
lung neoplasms, carcinoma in situ, typical carcinoid tumors, and
atypical carcinoid tumors. Lung cancer includes small cell lung
cancer ("SLCL"), non-small cell lung cancer ("NSCLC"), squamous
cell carcinoma, adenocarcinoma, small cell carcinoma, large cell
carcinoma, squamous cell carcinoma, and mesothelioma. Lung cancer
can include "scar carcinoma", bronchioloalveolar carcinoma, giant
cell carcinoma, spindle cell carcinoma, and large cell
neuroendocrine carcinoma. Lung cancer includes lung neoplasms
having histologic and ultrastructural heterogeneity (e.g., mixed
cell types).
[0169] As used herein, "cell proliferative diseases or disorders of
the colon" include all forms of cell proliferative disorders
affecting colon cells, including colon cancer, a precancer or
precancerous conditions of the colon, adenomatous polyps of the
colon and metachronous lesions of the colon. Colon cancer includes
sporadic and hereditary colon cancer. Colon cancer includes
malignant colon neoplasms, carcinoma in situ, typical carcinoid
tumors, and atypical carcinoid tumors. Colon cancer includes
adenocarcinoma, squamous cell carcinoma, and squamous cell
carcinoma. Colon cancer can be associated with a hereditary
syndrome such as hereditary nonpolyposis colorectal cancer,
familiar adenomatous polyposis, MYH-associated polyposis, Gardner's
syndrome, Peutz-Jeghers syndrome, Turcot's syndrome and juvenile
polyposis. Cell proliferative disorders of the colon can be
characterized by hyperplasia, metaplasia, and dysplasia of the
colon.
[0170] As used herein, "cell proliferative diseases or disorders of
the pancreas" include all forms of cell proliferative disorders
affecting pancreatic cells. Cell proliferative disorders of the
pancreas may include pancreatic cancer, an precancer or
precancerous condition of the pancreas, hyperplasia of the
pancreas, and dysplasia of the pancreas, benign growths or lesions
of the pancreas, and malignant growths or lesions of the pancreas,
and metastatic lesions in tissue and organs in the body other than
the pancreas. Pancreatic cancer includes all forms of cancer of the
pancreas, including ductal adenocarcinoma, adenosquamous carcinoma,
pleomorphic giant cell carcinoma, mucinous adenocarcinoma,
osteoclast-like giant cell carcinoma, mucinous cystadenocarcinoma,
acinar carcinoma, unclassified large cell carcinoma, small cell
carcinoma, pancreatoblastoma, papillary neoplasm, mucinous
cystadenoma, papillary cystic neoplasm, and serous cystadenoma, and
pancreatic neoplasms having histologic and ultrastructural
heterogeneity (e.g., mixed cell types).
[0171] As used herein, "cell proliferative diseases or disorders of
the prostate" include all forms of cell proliferative disorders
affecting the prostate. Cell proliferative disorders of the
prostate may include prostate cancer, a precancer or precancerous
condition of the prostate, benign growths or lesions of the
prostate, and malignant growths or lesions of the prostate, and
metastatic lesions in tissue and organs in the body other than the
prostate. Cell proliferative disorders of the prostate may include
hyperplasia, metaplasia, and dysplasia of the prostate.
[0172] As used herein, "cell proliferative diseases or disorders of
the skin" include all forms of cell proliferative disorders
affecting skin cells. Cell proliferative disorders of the skin may
include a precancer or precancerous condition of the skin, benign
growths or lesions of the skin, melanoma, malignant melanoma or
other malignant growths or lesions of the skin, and metastatic
lesions in tissue and organs in the body other than the skin. Cell
proliferative disorders of the skin may include hyperplasia,
metaplasia, and dysplasia of the prostate.
[0173] As used herein, "cell proliferative diseases or disorders of
the ovary" include all forms of cell proliferative disorders
affecting cells of the ovary. Cell proliferative disorders of the
ovary may include a precancer or precancerous condition of the
ovary, benign growths or lesions of the ovary, ovarian cancer, and
metastatic lesions in tissue and organs in the body other than the
ovary.
[0174] As used herein, "cell proliferative diseases or disorders of
the breast" include all forms of cell proliferative disorders
affecting breast cells. Cell proliferative disorders of the breast
may include breast cancer, a precancer or precancerous condition of
the breast, benign growths or lesions of the breast, and metastatic
lesions in tissue and organs in the body other than the breast.
[0175] In some embodiments, wherein the method entails use of a
bifunctional compound that targets a BRD protein, the subject may
have a cancer e.g., NUT midline carcinoma, treatment-refractory
acute myeloid leukemia, myelodysplastic syndrome, multiple myeloma,
triple negative- and estrogen receptor-positive breast cancers,
small cell and non-small cell lung cancers, castration resistant
prostate cancer, pancreatic ductal adenocarcinoma, colorectal
cancer, neuroblastoma and N-Myc Proto-Oncogene Protein
(MYCN)-driven solid tumors.
[0176] The compounds of the present application may be administered
to a patient, e.g., a cancer patient, as a monotherapy or by way of
combination therapy, and as a front-line therapy or a follow-on
therapy for patients who are unresponsive to front line therapy.
Therapy may be "first-line", i.e., as an initial treatment in
patients who have undergone no prior anti-cancer treatment
regimens, either alone or in combination with other treatments; or
"second-line", as a treatment in patients who have undergone a
prior anti-cancer treatment regimen, either alone or in combination
with other treatments; or as "third-line", "fourth-line", etc.
treatments, either alone or in combination with other treatments.
Therapy may also be given to patients who have had previous
treatments which have been partially successful but are intolerant
to the particular treatment. Therapy may also be given as an
adjuvant treatment, i.e., to prevent reoccurrence of cancer in
patients with no currently detectable disease or after surgical
removal of a tumor. Thus, in some embodiments, the compound may be
administered to a patient who has received another therapy, such as
chemotherapy, radioimmunotherapy, surgical therapy, immunotherapy,
radiation therapy, targeted therapy or any combination thereof.
[0177] The methods of the present application may entail
administration of compounds of the invention or pharmaceutical
compositions thereof to the patient in a single dose or in multiple
doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more doses).
For example, the frequency of administration may range from once a
day up to about once every eight weeks. In some embodiments, the
frequency of administration ranges from about once a day for 1, 2,
3, 4, 5 or 6 weeks, and in other embodiments entails a 28-day cycle
which includes daily administration for 3 weeks (21 days).
[0178] Combination Therapy
[0179] The compounds of the present invention may be used in
combination with at least one other active agent, e.g., anti-cancer
agent or regimen, in treating diseases and disorders. The term "in
combination" in this context means that the agents are
co-administered, which includes substantially contemporaneous
administration, by the same or separate dosage forms, or
sequentially, e.g., as part of the same treatment regimen or by way
of successive treatment regimens. Thus, if given sequentially, at
the onset of administration of the second compound, the first of
the two compounds is in some cases still detectable at effective
concentrations at the site of treatment.
[0180] In some embodiments, the treatment regimen may include
administration of a compound of the invention in combination with
one or more additional anticancer therapeutics. The dosage of the
additional anticancer therapeutic may be the same or even lower
than known or recommended doses. See, Hardman et al., eds., Goodman
& Gilman's The Pharmacological Basis Of Basis Of Therapeutics,
10th ed., McGraw-Hill, New York, 2001; Physician's Desk Reference
60th ed., 2006. Anti-cancer agents that may be used in combination
with the inventive compounds are known in the art. See, e.g., U.S.
Pat. No. 9,101,622 (Section 5.2 thereof). Representative examples
of additional active agents and treatment regimens include
radiation therapy, chemotherapeutics (e.g., mitotic inhibitors,
angiogenesis inhibitors, anti-hormones, autophagy inhibitors,
alkylating agents, intercalating antibiotics, growth factor
inhibitors, anti-androgens, signal transduction pathway inhibitors,
anti-microtubule agents, platinum coordination complexes, HDAC
inhibitors, proteasome inhibitors, and topoisomerase inhibitors),
immunomodulators, therapeutic antibodies (e.g., mono-specific and
bispecific antibodies) and CAR-T therapy.
[0181] In some embodiments, the compound of the invention and the
additional anticancer therapeutic may be administered less than 5
minutes apart, less than 30 minutes apart, less than 1 hour apart,
at about 1 hour apart, at about 1 to about 2 hours apart, at about
2 hours to about 3 hours apart, at about 3 hours to about 4 hours
apart, at about 4 hours to about 5 hours apart, at about 5 hours to
about 6 hours apart, at about 6 hours to about 7 hours apart, at
about 7 hours to about 8 hours apart, at about 8 hours to about 9
hours apart, at about 9 hours to about 10 hours apart, at about 10
hours to about 11 hours apart, at about 11 hours to about 12 hours
apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours
apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48
hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72
hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours
apart, or 96 hours to 120 hours part. The two or more anticancer
therapeutics may be administered within the same patient visit.
[0182] The active agents are administered concurrently to a subject
in the same or separate compositions. The combination therapeutics
of the invention may be administered to a subject by the same or
different routes of administration. The term "concurrently" is not
limited to the administration of the anticancer therapeutics at
exactly the same time. Rather, it is meant that they are
administered to a subject in a sequence and within a time interval
such that they can act together (e.g., synergistically to provide
an increased benefit than if they were administered otherwise). For
example, the therapeutics may be administered at the same time or
sequentially in any order at different points in time; however, if
not administered at the same time, they should be administered
sufficiently close in time so as to provide the desired therapeutic
effect, which may be in a synergistic fashion.
[0183] When the active components of the combination are not
administered in the same pharmaceutical composition, it is
understood that they can be administered in any order to a subject
in need thereof. For example, a compound of the present application
can be administered prior to (e.g., 5 minutes, 15 minutes, 30
minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours,
24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4
weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),
concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes,
30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12
hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3
weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the
administration of the additional anticancer therapeutic, to a
subject in need thereof. In various aspects, the anticancer
therapeutics are administered 1 minute apart, 10 minutes apart, 30
minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2
hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4
hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7
hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9
hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12
hours apart, no more than 24 hours apart or no more than 48 hours
apart. In one example, the anticancer therapeutics are administered
within the same office visit. In another example, the combination
anticancer therapeutics may be administered at 1 minute to 24 hours
apart.
[0184] In some embodiments, the compound of the present invention
and the additional agent or therapeutic (e.g., an anti-cancer
therapeutic) are cyclically administered. Cycling therapy involves
the administration of one anticancer therapeutic for a period of
time, followed by the administration of a second anti-cancer
therapeutic for a period of time and repeating this sequential
administration, i.e., the cycle, in order to reduce the development
of resistance to one or both of the anticancer therapeutics, to
avoid or reduce the side effects of one or both of the anticancer
therapeutics, and/or to improve the efficacy of the therapies. In
one example, cycling therapy involves the administration of a first
anticancer therapeutic for a period of time, followed by the
administration of a second anticancer therapeutic for a period of
time, optionally, followed by the administration of a third
anticancer therapeutic for a period of time and so forth, and
repeating this sequential administration, i.e., the cycle in order
to reduce the development of resistance to one of the anticancer
therapeutics, to avoid or reduce the side effects of one of the
anticancer therapeutics, and/or to improve the efficacy of the
anticancer therapeutics.
[0185] Pharmaceutical Kits
[0186] The present compositions may be assembled into kits or
pharmaceutical systems. Kits or pharmaceutical systems according to
this aspect of the invention include a carrier or package such as a
box, carton, tube or the like, having in close confinement therein
one or more containers, such as vials, tubes, ampoules, or bottles,
which contain the compound of the present application or a
pharmaceutical composition. The kits or pharmaceutical systems of
the invention may also include printed instructions for using the
compounds and compositions.
[0187] These and other aspects of the present application will be
further appreciated upon consideration of the following
non-limiting working examples.
EXAMPLES
[0188] By way of introduction, the Examples show that an inventive
compound ZXH-03-26 shows activity exclusively on the first
bromodomain of BRD4, and spares degradation of BRD2 or 3 at
concentrations >10 .mu.M (FIG. 6C), while non-inventive
bifunctional compounds dBET6 and MZ1 (used as controls) show
activity on most bromodomains (FIG. 6D). The examples also describe
experiments wherein bromodomain degradation for non-inventive
degrader dBET57 was assessed to test whether any short linker would
result in selectivity for BRD4.sub.BD1. In contrast to ZXH-03-26,
dBET57 is nearly equipotent on BRD3.sub.BD1 and BRD4.sub.BD1 (FIG.
6E). The examples describe further experiments designed to test
whether the selective ZXH-03-26 retains activity on endogenous full
length BRD4. HEK293T cells were treated with increasing
concentrations of ZXH-03-26. Immunoblot analysis confirms that
ZXH-03-26 degrades endogenous BRD4 with comparable efficacy
compared to the best pan-BET degrader dBET6 (FIG. 6G), while being
inactive on BRD2 and BRD3 (FIG. 6H).
[0189] Thus, the examples demonstrate that binding to a distinct
conformation can yield a highly selective degrader molecule and
that selectivity can be achieved across highly homologous domains
such as the bromodomains of BET proteins.
[0190] More specifically, the following Examples present a
comprehensive structural, biochemical and cellular analysis of
heterobifunctional compound (PROTAC)-mediated degradation of BET
family proteins, including specific degradation of BRD4 over other
BET family proteins. The Examples demonstrate that the
ligase-PROTAC-substrate binding mode is unexpectedly plastic, and
that this plasticity results in multiple low energy binding
conformations that can be exploited to achieve favourable binding
modes and help to rationalize heterobifunctional compound
specificity. Based on this new finding, heterobifunctional
compounds that are specific for BRD4 over other homologous BET
family proteins are synthesized, and specific degradation of BRD4
by these working examples of the invention are shown.
[0191] Through multiple X-ray crystal structures of PROTAC bound
CRL4.sup.CRBN-BRD4 complexes, the Examples below demonstrate that
plastic inter-protein contacts result in multiple distinct binding
conformations depending on the bound PROTAC. The Examples also
demonstrate that effective degradation does not require tight
cooperative binding; however, distinct binding conformations are
unique to ligase-substrate pairs and define selectivity. The
Examples further demonstrate a computational approach to
protein-protein docking and demonstrate the versatility of this
approach through rational design of the first PROTAC that can
discriminate between the highly homologous BET bromodomains of
BRD2/3/4, leading to synthesis of a highly effective and selective
BRD4 degrader.
[0192] The Examples provide a detailed understanding of the
molecular basis for target recruitment and selectivity, which is
critically required to enable rational design of degraders. The
Examples utilize comprehensive characterization of the ligand
dependent CRBN/BRD4 interaction to demonstrate that binding between
proteins that have not evolved to interact is unexpectedly plastic.
Multiple X-ray crystal structures show that plasticity results in
several distinct low energy binding conformations, which are
selectively bound by ligands. The Examples demonstrate that
computational protein-protein docking can reveal the underlying
inter-protein contacts and inform the design of BRD4 selective
degraders that can discriminate between highly homologous BET
bromodomains. The Examples demonstrating that plastic inter-protein
contacts confer selectivity for ligand-induced protein dimerization
provide a conceptual framework for the development of high
specificity heterobifunctional compounds. The Examples further
provide exemplary heterobifunctional compounds that are specific
for BRD4 over other BET family proteins.
[0193] Since small changes to the PROTAC can result in dramatically
altered cell permeability or solubility, the Examples below devised
a synthetic system based on the recruitment of isolated BRD4
bromodomains to CRL4.sup.CRBN. Like other members of the BET
family, BRD4 contains two bromodomains: bromodomain 1 (aa 75-147
and referred to as BRD4.sub.BD1) and BRD4.sub.BD2 (aa 368-440), and
sequence conservation between the two is limited (FIG. 7C-FIG. 7E).
These distinct domains bind the JQ1 based target-moiety with equal
affinities (Filippakopoulos, Qi et al. 2010), hence establish a
model system to understand how amino acid sequence and thereby
protein surface properties influence protein dimerization. The
Examples below utilized a series of compounds synthesized to bind
CRBN and the bromodomains of BRD4 (referred to as dBETs, see FIG.
7B) (Winter, Buckley et al. 2015). dBET molecules comprise the
E3-moiety thalidomide to bind to CRL4.sup.cRBN, a flexible linker
of variable length and composition, and a target-moiety, JQ1, that
binds to BRD4.sub.BD1 and BRD4.sub.BD2 with equal affinities
(Filippakopoulos, Qi et al. 2010).
Example 1: Crystal Structure of a
DDB1.DELTA.B-CRBN-dBET23-BRD4.sub.BD1 Complex
[0194] To determine the structural basis of BRD4 recruitment to
CRBN, DDB1.DELTA.B-CRBN, and BRD4.sub.BD1 complexes bound to
different dBET molecules were reconstituted. Initial crystals were
obtained for the .about.165 kDa
hsDDB1.DELTA.B-hsCRBN-dBET23-hsBRD4.sub.BD1 (dBET23 comprises an
8-carbon linker to bridge the oxy-acetamide of pomalidomide to the
thiophene group of JQ1) complex and its structure was determined to
3.5 .ANG. resolution (FIG. 1B) by molecular replacement using a
DDB1.DELTA.B-CRBN model (PDB: 5fqd, see Table 1). The DDB1
.beta.-propeller domains A and C (BPA and BPC) bind CRBN but do not
contribute contacts to BRD4.sub.BD1. CRBN consists of three
domains, the N-terminal domain (NTD), the helical-bundle domain
(HBD) and the C-terminal domain (CTD), which harbours the
thalidomide binding pocket (Fischer, Bohm et al. 2014). The small
molecule degrader dBET23 occupies the canonical binding sites on
CRBN and BRD4.sub.BD1 for lenalidomide and JQ1, respectively (FIG.
1C).
[0195] BRD4.sub.BD1 interacts with CRBN through contacts with the
NTD domain of CRBN and with CRBN residues in direct proximity to
the thalidomide-binding pocket (FIG. 1D). CRBN binds the
BRD4.sub.BD1 .alpha.C helix (aa 145-161) and residues in the
BRD4.sub.BD1 ZA loop (aa 76-104) (Filippakopoulos, Picaud et al.
2012). The .alpha.C helix forms hydrophobic interactions with two
loops in the CRBN-NTD (aa 101-104 and aa 147-154). Residues Leu148,
Met149, Ala152, and Leu156 in the .alpha.C helix together with
His77 and Phe79 in the ZA loop, form a hydrophobic patch that
interacts with Phe102, His103, Phe150, Gly151, Ile152, and Ile154
in the CRBN-NTD. BRD4.sub.BD1 Gln78 forms a hydrogen bond with
Gln100 in the CRBN-NTD (FIG. 1D). Consequently, mutations of the
BRD4.sub.BD1 residues Phe79Asp, Ala152Asp, and Gln78Ala all reduce
tertiary complex formation as monitored by measuring the
peak-height in a TR-FRET dimerization assay (FIG. 2A). The Examples
further showed that Asp145 is buried in a hydrophobic environment,
and accordingly, introducing an Asp145Ala mutation strengthens the
binding of BRD4.sub.BD1 to CRBN (FIG. 2A). The interaction between
CRBN and BRD4.sub.BD1 consists of a total buried surface area of
.about.550 .ANG..sup.2 (FIG. 2B) (Krissinel and Henrick 2007),
comparable to that observed for CRBN-Ck1.alpha. (.about.600
.ANG..sup.2) and GSPT1 (.about.600 .ANG..sup.2) (Matyskiela, Lu et
al. 2016, Petzold, Fischer et al. 2016).
[0196] In addition to dBET23, the Examples determined crystal
structure with the related molecules dBET6 (3.3 .ANG. resolution),
dBET70 (4.3 .ANG. resolution)--both have linkers of similar
length--and significantly longer dBET55 (4.0 .ANG. resolution and
crystallized with BRD4.sub.BD1 (D145A)). The overall structures of
these complexes are comparable to the structure obtained with
dBET23 (FIGS. 8A and B) and the involvement of near identical
inter-protein contacts is further confirmed by similar effects of
BRD4.sub.BD1 interface mutations on complex formation (FIG.
8C).
Example 2: Inter-Protein Contacts are Unique to BRD4.sub.BD1
[0197] The amino acid sequences of BRD4.sub.BD1 to BRD4B.sub.D2 are
49% similar (FIG. 7D), yet none of the key residues in the .alpha.C
helix or the ZA loop involved in contacts with CRBN are identical.
The Examples addressed whether affinity of BRD4.sub.BD2 for CRBN is
reduced in the presence of dBET6 or dBET23. While the determination
of absolute binding affinities is difficult for a three body
binding problem (Douglass, Miller et al. 2013), a qualitative
measure of the relative affinities (or cooperativity of binding)
can be indirectly obtained through CRBN-dBET binding assays in
presence or absence of purified BRD4.sub.BD1 or BRD4.sub.BD2
protein. Using a lenalidomide-Atto565 fluorescent probe, binding of
dBETs to CRBN was measured by competitive titration (FIGS. 2C-F).
Next, the Examples show similar binding experiments in presence of
increasing concentrations of either BRD4.sub.BD1 or BRD4.sub.BD2 to
assess the cooperativity of ternary complex formation. An apparent
cooperativity factor alpha was defined as
.alpha..sub.app=IC.sub.50[binary]/IC.sub.50[ternary], with positive
cooperativity resulting in .alpha..sub.app>1, and negative
cooperativity in .alpha..sub.app<1 (see FIG. 2C-FIG. 2F and FIG.
9A-FIG. 9G). dBET6, exhibited an IC.sub.50 of .about.0.8 .mu.M in
the absence of BRD4, which increases to an IC.sub.50 of 1.8 .mu.M
(.alpha..sub.app=0.6) in the presence of BRD4.sub.BD1, and an
IC.sub.50 of .about.4.1 .mu.M (.alpha..sub.app=0.2) in the presence
of BRD4.sub.BD2 (FIG. 2D and FIG. 9A-FIG. 9C), indicative of
negative cooperativity for both BRD4.sub.BD1 and BRD4.sub.BD2. For
dBET23 and dBET57 the difference between BRD4.sub.BD1 and
BRD4.sub.BD2 is more pronounced, with .alpha..sub.app=0.4 (dBET23)
and .alpha..sub.app=0.8 (dBET57) for BRD4.sub.BD1 and
.alpha..sub.app<0.1 for BRD4.sub.BD2 (the binding in presence of
BRD4.sub.BD2 is too weak to quantify), indicating negative
cooperativity and a preference for binding to BRD4.sub.BD1 (FIG. 2E
and FIG. 2F and FIG. 9A-FIG. 9G).
[0198] To better understand the drivers of selectivity and to test
whether the observed differences in cooperativity would result in
differential degradation of isolated BRD4 bromodomains, a system
was developed that allowed us to directly quantify cellular
degradation of either BRD4.sub.BD1 or BRD4.sub.BD2. Reporter cells
that stably express BRD4.sub.BD1-EGFP followed by a P2A splice site
separated mCherry, were treated with increasing concentrations of
dBET molecules (FIG. 3A-FIG. 3F). This assay format enables
quantitative readout of BRD4.sub.BD1 degradation with the
GFP/mCherry ratio using flow cytometry (similar reporter cells were
used for BRD4.sub.BD2, or an IKZF protein that has internal
deletions 41-82, 4197-239, and 4256-519 hereafter referred to as
IKZF.DELTA.). The Examples demonstrate that dBET6
(DC.sub.50/5h.about.10 nM, with DC.sub.50/5h referring to
half-maximal degradation after 5 hours of treatment), dBET23
(DC.sub.50/5h.about.50 nM) and dBET70 (DC.sub.50/5h.about.5 nM)
exhibit the most potent effects on BRD4.sub.BD1 protein levels,
followed by dBET1 (DC.sub.50/5h.about.500 nM) and dBET57
(DC.sub.50/5h.about.500 nM) (FIGS. 3A-C and FIGS. 10A-L). For
BRD4.sub.BD2, dBET70 (DC.sub.50/5h.about.5 nM) has the most
pronounced effects, followed by dBET6 (DC.sub.50/5h.about.50 nM),
dBET23 (DC.sub.50/5h>1 .mu.M) and dBET1 (DC.sub.50/5h dBET57,
which exhibits significant degradation of BRD4.sub.BD1, is inactive
on BRD4.sub.BD2 (FIGS. 3D-F and FIGS. 10A-L). The cellular activity
is thus directly proportional to the observed cooperativity factors
(FIGS. 9A-B), and dBET57 was found remarkably selective for
BRD4.sub.BD1 in biochemical and cellular assays (FIG. 2F and FIGS.
3A-F).
Example 3: Plastic Binding Confers Selectivity to dBETs
[0199] When comparing the CRBN-dBET23-BRD4.sub.BD1 structure to the
previously determined structures of CRBN-Ck1.alpha. (Petzold,
Fischer et al. 2016), and CRBN-GSPT1 (Matyskiela, Lu et al. 2016),
the Examples show that these neo-substrates use different surfaces
on CRBN to stabilize tertiary complex formation (FIG. 11A). The
Examples also show that molecules with short linkers, such as
dBET57, would not be able to dimerize CRBN and BRD4 in the
conformation observed in the CRBN-dBET23-BRD4.sub.BD1 structure
since a minimum of 8 carbons would be required to bridge the
E3-moeity with the target-moiety and dBET57 comprises a 2-carbon
linker (FIG. 11C). Additional Examples address whether dBET
molecules incompatible with the observed binding mode, such as
dBET57 or dBET1, would bind in a different overall
conformation.
[0200] To explore potential differences in binding, mutational
analysis was performed. A set of single amino acid point mutations
was introduced in CRBN and BRD4.sub.BD1 to obtain a mutational
signature of binding. When comparing the mutational signatures of
different dBETs, the Examples show that while dBET6 and 23 share
similar profiles (FIGS. 4A and B, and 11D and E), the mutational
signatures of dBET1 and dBET57 are distinct (FIG. 4A-FIG. 4D and
FIG. 11D-FIG. 11I). This suggests that different dBET
molecules--depending on linker length and linkage position--result
in distinct binding conformations of CRBN-BRD4 complex
formation.
[0201] To obtain insights into the molecular basis of this plastic
CRBN/BRD4.sub.BD1 interactions, dBET57 (the molecule with the most
pronounced selectivity for BRD4.sub.BD1 over BRD4.sub.BD2.) was
crystallized. Crystals were obtained for a reconstituted
DDB1.DELTA.B-CRBN-dBET57-BRD4.sub.BD1 complex and determined the
structure to 6.8 .ANG. resolution (see FIG. 12A-FIG. 12C for
experimental validation of the structure). While the limited
resolution prevents detailed interpretation of the molecular
interactions that govern the CRBN-BRD4 interface, the overall
binding mode is clearly resolved (FIG. 4F and FIG. 12A). In this
complex, BRD4.sub.BD1 interacts with the CTD of CRBN, instead of
the NTD as observed with dBET6/23 (FIG. 4E-FIG. 4H), which results
in BRD4 now utilizing an entirely different set of residues for
inter-protein contexts (compare FIG. 2B and FIG. 4H). In the dBET57
bound structure, the Examples show that CRBN unfolds and the
CRBN-NTD and CRBN-CTD domains no longer interact (FIG. 4E-FIG. 4F).
This unexpected behaviour could be due to the high salt
crystallization condition (1.6 M Phosphate) or part of the
intrinsic CRBN plasticity. The binding mode observed with dBET57,
however, is fully compatible with a regular CRBN conformation (FIG.
4G) and dBET57 mediated binding thus expected to occur with both
CRBN conformations (see FIG. 12A-FIG. 12C). The unexpected
plasticity in dBET dependent binding of CRBN to the exact same
protein, BRD4.sub.BD1, provides a rationale how PROTACs that share
the same E3- and target-moieties can still exhibit different
selectivity profiles. Depending on the linker, different surface
residues in the target protein may be involved in complex
formation.
[0202] FIG. 12A shows that CRBN was found in a not previously
observed conformation, in which the thalidomide binding CRBN-CTD
domain translates and rotates away from the CRBN-HBD and CRBN-NTD
domains. This results in an open conformation that exposes large
areas of CRBN that are typically buried. The high salt
crystallization condition could be a driver of this structural
rearrangement, and together with crystal contacts induce this
conformation. It is possible that that this conformational dynamic
is an intrinsic feature of CRBN to accommodate a variety of
substrates and future studies are necessary to address this. Based
on the compatibility of the observed BRD4.sub.BD1 binding
conformation with the open and closed CRBN conformations, it can be
concluded that for the interpretation of the data, the
conformational change is negligible.
Example 4: Protein Docking Reveals Binding Energy Landscape
[0203] The mutational signatures obtained for different dBET
molecules, the structural arrangements for dBET6/23/70 and dBET57
complexes, together with the absence of any co-evolution between
CRBN and BRD4 let us hypothesize that BRD4 bromodomains can bind to
CRBN in multiple different orientations depending on the ligand.
Assessing such potential binding conformations to reduce chemical
search space would be highly desirable. In silico protein-protein
docking provides an attractive surrogate to in vitro experiments.
The Examples addressed whether the Rosetta protein docking
framework (Sircar, Chaudhury et al. 2010) would allow modelling of
such possible binding modes. One of the characteristics of
Monte-Carlo docking algorithms is the stochastic sampling of low
energy conformations, which frequently results in multiple
solutions. While this often complicates the identification of
evolved interactions between proteins, sampling of possible
conformations provides an advantage in the study of
degrader-induced binding modes since it enables exploration of the
repertoire of low energy conformations.
[0204] The Examples confirmed that computational methods can
predict ligand mediated protein-protein interactions by docking
Ck1.alpha. to the CRBN-lenalidomide complex (FIG. 13A-FIG. 13D).
The Examples further addressed whether computational docking would
be able to provide models for possible PROTAC-induced binding modes
by docking CRBN and the target BRD4.sub.BD1 in absence of dBET. One
obvious complication is that a dominant component of the binding
energy between ligase and substrate is provided by the degrader
itself, which is absent in docking simulations, and the scoring of
solely neomorphic interactions will likely result in many low
energy conformations to be generated.
[0205] Using the crystal structure of lenalidomide bound CRBN (pdb:
4tz4) and JQ1 bound BRD4.sub.BD1 (pdb: 3mxf), a global docking
experiment (20,000 models) was performed using Rosetta docking
(FIG. 5A). Clustering the top 200 lowest scoring docking
conformations, a conformation was identified that closely resembles
the conformation observed in the dBET23 crystals. This model was
further confirmed by local docking (2,000 models) of the low energy
model (FIG. 5A and FIG. 5B).
[0206] As predicted for a much weaker interaction between CRBN and
BRD4.sub.BD1 in absence of a degrader, multiple low energy minima
are found. Based on the hypothesis that the docking experiment will
sample the repertoire of low energy binding conformations,
clustering of the top 200 conformations provides a set of feasible
binding modes (see, FIG. 5C) for representative clusters). While it
remains to be shown whether docking can predict binding modes
accurately, the overall conformational landscape provides a
rationale for the design of required minimal linker lengths and
suggest suitable linkage positions. In theory, the shortest
possible linker for a ligase-target pair should provide the most
selective compound since it will restrict the number of possible
binding conformations. To test whether the docking information
could be used to inform the design of PROTACs, poses were sorted by
minimal required linker length between the JQ1 thiophene and
lenalidomide, and found a linker of 2-3 atoms sufficient to bridge
the two moieties (FIG. 6A). The according molecules (ZXH-02-147 and
ZXH-03-26) were synthesized (FIGS. 6B and 7B).
[0207] The Examples addressed whether certain degraders (PROTACs)
would be capable of directly inducing binding of IKZF1 (and other
IMiD targets) to CRBN. A CRBN-IKZF1.DELTA. binding assay was used
to measure binding of IKZF1.DELTA., to CRBN in presence of dBET1,
dBET6, dBET23, dBET57, dBET70, and dBET72, as well as lenalidomide
as control (FIG. 14A). The Examples show that dBET1/6/23 do not
induce IKZF1-CRBN complex formation, while dBET57, dBET70 and
dBET72 show pronounced complex formation. Both, dBET57 and dBET70
share the aniline of lenalidomide, while dBET1/6/23 all have an
oxy-acetamide linkage. Based on the previously described model of
IKZF1-CRBN binding (FIG. 14C) the phthalimide aniline nitrogen may
be involved in a hydrogen bond with IKZF1 Q146. A straight linker
out of this phthalimide position could be tolerated, while an
adjacent amide bond (as in the oxy-acetamide linkage) may cause a
steric clash with IKZF1. Alternatively, the secondary amine
nitrogen could be a hydrogen bond donor and, with the ether oxygen
being a hydrogen bond acceptor, this donor/acceptor substitution
could explain the difference in strength of the IKZF1 interaction.
The nitrogen linkage of dBET57, dBET70 and dBET72 were replaced
with an oxygen-ether linkage resulting in compounds ZXH-2-42,
ZXH-2-43, and ZXH-2-45, respectively. The ability of the
oxygen-ether compounds to induce binding of IKZF1 was greatly
reduced compared to their nitrogen analogs; however, it was not
eliminated, as seen in the case of the oxy-acetamide
substitution.
Example 5: Dose Dependent Degradation of an IKZF1.DELTA.-EGFP
Fusion Protein
[0208] Dose dependent degradation of an IKZF1.DELTA.-EGFP fusion
protein was assessed in HEK293T cells (see methods), and used the
in vitro structure activity relationship (SAR) to develop a model
of cellular IKZF1 degradation (FIG. 14B). dBET1/6/23 are relatively
ineffective at promoting IKZF1 degradation, dBET70/72 are
equipotent to lenalidomide, and dBET57 is comparable to
thalidomide, in accordance with the biochemical data. The Examples
show that by modifying the substitution at the IMiD moiety, the
co-degradation of other substrates--such as IKZF1--can be
controlled or modulated. To test whether this would be effective in
a cellular multiple myeloma model, MM.1s cells were treated for
five hours with either 1 .mu.M dBET23, 1 .mu.M dBET70 or DMSO as a
control. Using a quantitative proteomics approach (see methods),
the Examples demonstrate that dBET70 but not dBET23 exhibits
pronounced co-degradation of CRBN-lenalidomide neo-substrates
IKZF1, IKZF3 and ZFP91 (FIGS. 14D and E).
[0209] Cellular degradation assays show that ZXH-02-147 and
ZXH-03-26 are active on BRD4Bm, in accordance with the docking
results (FIGS. 6C and 15A), and that ZXH-03-26 exhibits a
DC.sub.50/5h.about.5 nM comparable to the best pan-BRD degrader
dBET6. To test whether these molecules exhibit isoform selectivity,
the cellular reporter system was expanded to include the individual
bromodomains of BRD2 and BRD3 and tested cellular degradation along
with BRD4.
Discussion of Examples 1-5
[0210] An integrated approach combining structural, biochemical,
and cellular data was used to establish the molecular basis of
PROTAC-mediated neo-substrate recruitment to the CRL4.sup.CRBN E3
ubiquitin ligase. The Examples above show that inter-protein
contacts, while contributing relatively little binding affinity to
the interaction, can be drivers of selectivity, and that highly
effective degraders (e.g. the low nanomolar (nM) cellular activity
of dBET6 or dBET70) can be achieved in absence of tight binding or
positive cooperativity. Through multiple X-ray crystal structures
together with comprehensive biochemical, cellular, and
computational characterization, the Examples demonstrate that
binding between ligase and substrate is surprisingly plastic and
thus adapt distinct conformations depending on linker length and
position. The Examples also demonstrate that exploiting such
`local` energy/entropy minima underlies selectivity as seen for
dBET57. The Examples further demonstrate that in silico protein
docking can be used to reveal low energy binding modes and can
guide development of heterobifunctional degraders that can
discriminate between the highly homologous BET bromodomains, such
as ZXH-03-26. The Examples above further demonstrate that
biochemical properties translate to cellular activity with respect
to BRD4 on-target and IKZF1 off-target degradation and that the
IKZF1 degradation can be tuned by IMiD linker composition (FIG.
14A-FIG. 14E).
[0211] The Examples above demonstrate that the same two proteins
can bind in different overall conformations, which results in
distinct surface patches on the ligase and target to interact. This
plasticity underlies the principle of selectivity. PROTACs
therefore appear to exploit natural and widely occurring
non-specific interactions by increasing the local concentration of
the two protein binding partners. Non-specific interactions are
widespread and thought to occur between any two proteins with
affinities >10 mM (Kuriyan and Eisenberg 2007). However, these
interaction surfaces are not random as they require a certain
degree of surface complementarity to avoid unfavourable contacts
such as opposing charged surfaces. The constraints of relatively
short linkers result in only few accessible inter-protein contact
conformations. In theory, rationally designed linkers restricted to
a specific binding mode unique to a ligase/substrate pair should be
sufficient to drive selectivity since such a restricted
conformation is unlikely to occur in a close orthologue. The
Examples above show that such can be achieved in practice with the
compound ZXH-03-26.
[0212] The absence of positive cooperativity and the existence of
multiple distinct binding conformations carries further important
implications. The unnecessity for high affinity ligase-substrate
interactions implies that a wide variety of E3 ligases can be
explored to achieve desirable properties such as tissue
specificity. The Examples above demonstrate with dBET57 and
ZXH-03-26 that effective PROTACs can be designed to harbour
relatively short linkers, which results in favourable and more
`drug-like` overall properties (FIG. 7B). The Examples above
demonstrate that such short linker compounds exhibit high
selectivity since the number of accessible binding conformations is
reduced. Selectivity can also be further explored using different
E3-moeities, as seen for CRBN- and VHL-targeting PROTACs (FIGS.
3A-C). The Examples above demonstrate that computational modelling
can provide an elegant surrogate, which depends only on a known
structure for the individual components (ligase and target), and
has the potential to enable initial predictions of possible linker
length and trajectory to guide medicinal chemistry.
[0213] With ZXH-03-26, ZXH-2-184, ZXH-2-147, and ZXH-3-82, the
Examples above provides working examples of heterobifunctional
compounds that selectively targets BRD4 for degradation and spares
BRD2 and BRD3, which also represents the first small molecule to
allow pharmacologic targeting of BRD4 without significant
inhibition/degradation of BRD2/3. This has implications for future
developments since efficacy of BRD4 inhibition has been established
for a variety of malignancies (Zuber, Shi et al. 2011, Chau,
Hurwitz et al. 2016), while on-target toxicity has been observed in
pre-clinical and clinical studies (Stathis, Zucca et al. 2016). It
is conceivable that selective degradation of BRD4 will retain
efficacy, while significantly reducing on-target toxicity in NUT
midline carcinomas, which depend on the BRD4-NUT fusion protein.
Such selective targeting of an oncogenic fusion protein has been
shown as effective treatment strategy in the case of BCR-ABL and
Gleevec (Buchdunger, Cioffi et al. 2000). ZXH-03-26, ZXH-2-184,
ZXH-2-147, and ZXH-3-82 present examples of heterobifunctional
compounds that can selectively degrade the BRD4-NUT oncogenic
fusion protein.
Example 6: Cellular Imaging-Based Degradation Assay
[0214] Close analogs of ZXH-03-26 were further explored using
cellular imaging-based assay (FIG. 19-FIG. 20) Substitution in
linker composition from secondary amine nitrogen (as in ZXH-3-26)
to oxygen (as in BJG-02-030) maintained BRD4.sub.BD1 degradation
selectivity, with reduced activity (FIG. 19A). In addition,
location of fluorine substitution in pthalimide ring of IMiD, has
shown to be critical with BJG-02-119 maintaining selectivity, with
reduced activity as compared to ZXH-3-26, and BJG-01-174 resulting
in inactive degrader (FIG. 19A-FIG. 19G). Furthermore, more rigid
oxoacetamide linker analog of ZXH-3-26, results in inactive
degrader ZXH-4-28, changing the linker exit position on IMiD as in
ZXH-3-28, also results in inactive molecule, suggesting that both
the linker attachment chemistry and the attachment location are
crucial in maintaining active degradation. BRD3/BRD4 selective
degraders were also observed as exemplified by ZXH-3-52 (FIG. 19 D)
and to lesser extend ZXH-3-195 (FIG. 19 E).
[0215] Further increasing ZXH-3-26 linker length by one atom
results in loss of selectivity as observed for ZXH-3-117 (FIG.
20A-FIG. 20D). Short ether linker analog ZXH-2-42 showed
significantly reduced activity.
[0216] Finally, as shown on FIGS. 14A and 14B, compounds ZXH-2-43
and ZXH-2-45 that showed reduced IKZF1 binding and IKZF1
degradation were able to induce potent degradation of bromodomains.
Compound ZXH-2-43 showed significant degradation of BRD2/3/4 even
at 2.6 nM concentration (FIG. 20 C).
[0217] Cells stably expressing bromodomain-GFP with mCherry
reporter were seeded at 30-50% confluency in 384 well plates (3764,
Corning) with 50 .mu.L FluoroBrite DMEM media (Gibco, A18967)
containing 10% FBS per well a day before compound treatment.
Compounds (see Figure legends) were dispensed using D300e Digital
Dispenser (HP) normalized to 0.5% DMSO and incubated with cells for
5 h. The assay plate was imaged immediately using Acumen eX3/HCl
(TTPLabtech) High Content Imager with 488 nm and 561 nm lasers in 2
.mu.m.times.1 .mu.m grid per well format. The resulting images were
analyzed using CellProfiler.TM. (Carpenter, et al., GenomeBiology
7:r100 (2006)). A series of image analysis steps (`image analysis
pipeline`) was constructed.
[0218] The CellProfiler.TM. pipeline steps are briefly outlined
here. First, the red and green channels were aligned and cropped to
target the middle of each well (to avoid analysis of heavily
clumped cells at the edges), and a background illumination function
was calculated for both red and green channels of each well
individually and subtracted to correct for illumination variations
across the 384-well plate from various sources of error. An
additional step was then applied to the green channel to suppress
the analysis of large auto fluorescent artifacts and enhance the
analysis of cell specific fluorescence by way of selecting for
objects under a given size, 30 A.U., and with a given shape,
speckles. mCherry-positive cells were then identified in the red
channel filtering for objects between 8-60 pixels in diameter and
using intensity to distinguish between clumped objects. The green
channel was then segmented into GFP positive and negative areas and
objects were labeled as GFP positive if at least 40% of it
overlapped with a GFP positive area. The fraction of GFP-positive
cells/mCherry-positive cells (GFP/mCherry ratio) in each well was
then calculated, and the green and red images were rescaled for
visualization. The GFP/mCherry ratio was normalized to DMSO and
analyzed in GraphPad Prism 7.
Example 7: Constructs and Protein Purification
[0219] Wild-type and mutant versions of human DDB1, human CRBN, and
human IKZF1.DELTA. were cloned in pAC-derived vectors (Abdulrahman,
Uhring et al. 2009) and recombinant proteins were expressed as
N-terminal His.sub.6(DDB1.DELTA.B, CRBN), StrepII-Avi (IKZF1A) or
his.sub.6-3C-Spy (CRBN) (Zakeri, Fierer et al. 2012) fusions in
Trichoplusia ni High-Five insect cells using the baculovirus
expression system (Invitrogen). Wild-type and mutant BRD4.sub.BD1
and BRD4.sub.BD2 subcloned into E. coli pET100/D-TOPO vector with
N-terminal His.sub.6-Avi fusions were obtained from Invitrogen,
BRD4.sub.BD1/2 were subcloned into N-terminal his.sub.6-MBP-TEV-Spy
pETDuet vector and all expressed in BL21-DE3 or BL21-DE3 Rosetta
cells using standard protocols. For purification of His.sub.6 and
GST tagged proteins, cells were resuspended in buffer containing 50
mM tris (hydroxymethyl)aminomethane hydrochloride (Tris-HCl) pH
8.0, 200 mM NaCl, 1 mM tris (2-carboxyethyl)phosphine (TCEP), 1 mM
phenylmethylsulfonyl fluoride (PMSF), 1.times. protease inhibitor
cocktail (Sigma) and lysed by sonication. Cells expressing
StrepII-Avi-IKZF1A were lysed in the presence of 50 mM Tris-HCl pH
8.0, 500 mM NaCl, 1 mM TCEP, 1 mM PMSF and 1.times. protease
inhibitor cocktail (Sigma). Following ultracentrifugation, the
soluble fraction was passed over appropriate affinity resin
Strep-Tactin Sepharose (IBA) or Ni Sepharose 6 Fast Flow affinity
resin (GE Healthcare) or Glutathione Sepharose 4B (GE Healthcare)
and eluted with wash buffer (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 1
mM TCEP) supplemented with 2.5 mM D-Desthiobiotin (IBA) or 100 mM
imidazole (Fischer Chemical) or 10 mM glutathione (Fischer
BioReagents) respectively. The affinity-purified protein was either
further purified (CRBN-DDB1.DELTA.B, IKZF1A, Spy-BRD4.sub.BD1) via
ion exchange chromatography (Poros 50HQ) and subjected to size
exclusion chromatography or concentrated and directly loaded on the
size exclusion chromatography in 50 mM HEPES pH 7.4, 200 mM NaCl
and 1 mM TCEP. Biotinylation of IKZF1.DELTA. and BRD4.sub.BD1,
BRD4.sub.BD2 variants was performed as previously described
(Petzold, Fischer et al. 2016).
[0220] The protein-containing fractions were concentrated using
ultrafiltration (Millipore) and flash frozen in liquid nitrogen
(DDB1.DELTA.B-CRBN constructs at 40-120 .mu.M, biotinylated
His.sub.6-Avi-BRD4 mutants and WT, and not biotinylated WT at
.about.25-100 .mu.M, biotinylated StrepII-Avi-IKZF1 at .about.20
.mu.M concentration) and stored at -80.degree. C. or directly
covalently labelled with BODIPY-FL-SpyCatchers.sub.50c
(His.sub.6-3C-Spy-CRBN-His.sub.6-DDB1.DELTA.B, Spy-BRD4.sub.BD1) as
described below.
Example 8: Labelling of Spycatcher with BODIPY-FL-Maleimide
[0221] Spycatcher containing a Ser50Cys mutation was obtained as
synthetic dsDNA fragment from IDT (Integrated DNA technologies) and
subcloned as GST-TEV fusion protein in a pET-Duet derived vector.
Spycatcher S50C was expressed in BL21 DE3 and cells were lysed in
the presence of 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM TCEP and 1
mM PMSF. Following ultracentrifugation, the soluble fraction was
passed over Glutathione Sepharose 4B (GE Healthcare) and eluted
with wash buffer (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM TCEP)
supplemented with 10 mM glutathione (Fischer BioReagents). The
affinity-purified protein was subjected to size exclusion
chromatography, concentrated and flash frozen in liquid
nitrogen.
[0222] Purified Spycatchers.sub.50C protein was incubated with DTT
(8 mM) at 4.degree. C. for 1 h. DTT was removed using a ENRich
SEC650 10/300 (Bio-rad) size exclusion column in a buffer
containing 50 mM Tris pH 7.5 and 150 mM NaCl, 0.1 mM TCEP.
BODIPY-FL-maleimide (Thermo Fisher) was dissolved in 100% DMSO and
mixed with Spycatchers.sub.50C to achieve 2.5 molar excess of
BODIPY-FL-maleimide. SpyCatchers.sub.50C labelling was carried out
at room temperature (RT) for 3 h and stored overnight at 4.degree.
C. Labelled Spycatchers.sub.50C was purified on a ENRich SEC650
10/300 (Bio-rad) size exclusion column in 50 mM Tris pH 7.5, 150 mM
NaCl, 0.25 mM TCEP and 10% (v/v) glycerol, concentrated by
ultrafiltration (Millipore), flash frozen (.about.40 .mu.M) in
liquid nitrogen and stored at -80.degree. C.
Example 9: BODIPY-FL-Spycatcher Labelling of CRBN-DDB1.DELTA.B and
BRD4.sub.BD1
[0223] Purified His.sub.6-DDB1.DELTA.B-His.sub.6-3C-Spy-CRBN or
His.sub.6-Spy-BRD4.sub.BD1 was incubated overnight at 4.degree. C.
with BODIPY-FL labelled SpyCatchers.sub.50C protein at
stoichiometric ratio. Protein was concentrated and loaded on the
ENrich SEC 650 10/300 (Bio-rad) size exclusion column and the
fluorescence monitored with absorption at 280 and 490 nm. Protein
peak corresponding to the labeled protein was pooled, concentrated
by ultrafiltration (Millipore), flash frozen (.about.9.6 .mu.M for
His.sub.6-DDB1.DELTA.B-His.sub.6-3C-Spy-CRBN.sub.BODIPY SpyCatcher
or .about.22 uM for His.sub.6-Spy-BRD4.sub.BD1) in liquid nitrogen
and stored at -80.degree. C.
Example 10: Crystallization and Data Collection
[0224] Previously developed DDB1 construct was used that lack WD40
propeller B (BPB, residues 396-705) domain (Petzold, Fischer et al.
2016) (referred to as DDB1.DELTA.B) successful in crystallization
of lenalidomide-CK1.alpha. complex. For crystallization of
His.sub.6-DDB1.DELTA.B-His.sub.6-CRBN-dBET6/23/70-his.sub.6-BRD4.sub.BD1
and
His.sub.6-DDB1.DELTA.B-His.sub.6-CRBN-dBET55-His.sub.6-Avi-BRD4.sub.B-
D1 D145A complexes 145 .mu.M of dBET was mixed with 70 .mu.M
BRD4.sub.BD1 or BRD4.sub.BD1 D145A and 80 .mu.M
His.sub.6-DDB1.DELTA.B-His.sub.6-CRBN and incubated for 15 min
either on ice or at RT. Crystallisation plates were set up in 3
sub-well plates (Intelli, Art Robbins) by vapour diffusion using
NT8 (Formulatrix) at 20.degree. C. and images acquired using
Rocklmager.RTM. 1000 (Formulatrix.RTM.). Crystals appeared in wells
B9-F9 and H9 of Morpheus.RTM. HT Screen (Molecular Dimensions)
within few hours and were fully grown after 3 days. Single uniform
crystals (length 80-100 .mu.m) were present in condition C9 (10%
(w/v) PEG20k, 20% (w/v) PEG550 MME, 0.1 M BICINE pH 8.5) in 2:1 or
1:1 protein to precipitant ratio in 150 or 225 nL drops. Further
optimisation of condition in Morpheus.RTM. HT Screen C.sub.9 by
Silver Bullets (Hampton Research) additive screening in 1:10
additive to reservoir ratio resulted in optimal crystals for dBET6,
dBET23, dBET55 and dBET70 in Silver Bullet wells D7, B5, G4 and F6
respectively, in 2:1 protein to precipitant ratio of 225 or 400 nL
drops. Crystals were cryo-protected in reservoir solution
supplemented with 25-30% PEG 400 containing 150-300 .mu.M
respective dBET and flash-cooled in liquid nitrogen. The Examples
show that crystals harvested after 2-3 days resulted in optimal
diffraction. Diffraction data were collected at the APS Chicago
(beamline 24-ID-C) with a Pilatus 6M-F detector at a temperature of
100 K, or for dBET6 co-crystal structure at beamline 24-ID-E with a
Eiger 16M detector at a temperature of 100 K. Data were indexed and
integrated using XDS (Kabsch 2010) and scaled using AIMLESS
supported by other programs of the CCP4 suite (Winn, Ballard et al.
2011) or RAPD pipeline (APS Chicago). Data processing statistics,
refinement statistics and model quality parameters are provided in
Table 1.
[0225] dBET57 containing crystals were obtained by mixing
His.sub.6-DDB1.DELTA.B-His.sub.6-CRBN at 75 .mu.M, with dBET57 at
140 .mu.M and BRD4.sub.BD1 at 140 .mu.M in condition B5 of the
Hampton Index HT screen (1.26 M NaH.sub.2PO.sub.4, 0.14 M
K.sub.2HPO.sub.4). Single crystals were harvested, stabilized by
addition of 25% ethylene glycol containing dBET57 at 50 .mu.M.
Diffraction data were collected at the APS Chicago (beamline
24-ID-C) with a Pilatus 6M-F detector at a temperature of
100.degree. K, at wavelengths of 0.9962 .ANG. for native, 1.2828
.ANG. for Zn peak, and 1.7712 for S peak. Data were indexed and
integrated using XDS (Kabsch 2010) and scaled using AIMLESS
supported by other programs of the CCP4 suite (Winn, Ballard et al.
2011). Data processing statistics, refinement statistics and model
quality parameters are provided in Table 2.
Example 11: Structure Determination and Model Building
[0226] The DDB1.DELTA.B-CRBN-dBET6/23/70-BRD4.sub.BD1 and
DDB1.DELTA.B-CRBN-dBET55-BRD4.sub.BD1/D145A quaternary complexes
crystallized in space group P6.sub.522 with single complex in the
unit cell. PHASER (McCoy, Grosse-Kunstleve et al. 2007) was used to
determine the structures by molecular replacement using a
crystallographic model of DDB1.DELTA.B-CRBN omitting Ck1.alpha.
based on a crystal structure PDB 5fqd. The initial model was
iteratively improved with COOT and refined using PHENIX.REFINE
(Afonine, Grosse-Kunstleve et al. 2012) and autoBUSTER (Bricogne G,
Blanc E et al. 2011) with ligand restraints generated by Grade
server (Global Phasing) or phenix.elbow (Moriarty, Grosse-Kunstleve
et al. 2009). Protein geometry analysis revealed 0.63%, 0.55%,
0.94%, 0.72%, 1.02% Ramachandran outliers, with 95.43%, 95.27%,
94.68%, 93.99, 92.18% residues in favoured regions and 3.94%,
4.18%, 4.38%, 5.29%, 6.80% residues in allowed regions for the
complexes with dBET6, 23, 55, 57 and 70 respectively.
[0227] The DDB1.DELTA.B-CRBN-dBET57-BRD4.sub.BD1 complex
crystallized in space group 1422 with a single complex in the unit
cell. PHASER (McCoy, Grosse-Kunstleve et al. 2007) was used for
molecular replacement using models of hsDDB1.DELTA.B-hsCRBN-HBD
derived from pdb: 5fqd, hsCRBN-NTD derived from pdb: 5fqd, and
BRD4.sub.BD1 (pdb: 3mxf). The model was rigid body refined using
PHENIX.REFINE (Afonine, Grosse-Kunstleve et al. 2012) and the
hsCRBN-CTD was subsequently placed using Coot Jiggle-Fit (part of
Coot EM scripts from Alan Brown and Paul Emsley). The final model
was rigid body refined using PHENIX.REFINE and autoBUSTER (Bricogne
G, Blanc E et al. 2011). Anomalous maps were calculated with
PHENIX.MAPS (Afonine, Grosse-Kunstleve et al. 2012).
[0228] Figures were generated with PyMOL (The PyMOL Molecular
Graphics System, Version 1.8.6.0 Schrodinger, LLC) and model
quality was assessed with MOLPROBITY (Chen, Arendall et al. 2010).
Interaction surfaces were determined with PISA (Krissinel and
Henrick 2007). The IKZF1 homology model was taken from (Petzold,
Fischer et al. 2016).
Example 12: Time-Resolved Fluorescence Resonance Energy Transfer
(TR-FRET)
[0229] Compounds in dimerization assays were dispensed in a
384-well microplate (Corning, 4514) using D300e Digital Dispenser
(HP) normalized to 2% DMSO into 200 nM biotinylated
His.sub.6-avi-bromodomain (WT or mutant) or 80 nM biotinylated
Strepll-avi-IKZF1A, 100 nM
His.sub.6-DDB1.DELTA.B-His.sub.6-CRBN.sub.BODIPYY-Spycatcher and 2
nM terbium-coupled streptavidin (Invitrogen) in a buffer containing
50 mM Tris pH 7.5, 100 mM NaCl, 0.1% Pluronic.RTM. F-68 solution
(Sigma) and 2% DMSO (4% DMSO final). Compounds in CRBN mutants
dimerization assay were dispensed as described above into 200 nM
His.sub.6-DDB1-His.sub.6-CRBN.sub.mutants or 200 nM
His.sub.6-DDB1.DELTA.B-His.sub.6-CRBN.sub.WT, 100 nM
BRD4.sub.BD1-BODIPY-SpyCatcher and 2 nM terbium-anti-HIS Ab
(Invitrogen) in a buffer containing 50 mM Tris pH 7.5, 100 mM NaCl,
0.1% Pluronic F-68 solution (Sigma) and 2% DMSO (4% DMSO final).
Before TR-FRET measurements were conducted, the reactions were
incubated for 15 min at RT. After excitation of terbium
fluorescence at 337 nm, emission at 490 nm (terbium) and 520 nm
(BODIPY) were recorded with a 70 .mu.s delay over 600 .mu.s to
reduce background fluorescence and the reaction was followed over
30 200 second cycles of each data point using a PHERAstar.RTM. FS
microplate reader (BMG Labtech). The TR-FRET signal of each data
point was extracted by calculating the 520/490 nm ratio. The
heterobifunctional nature of small molecule degraders results in a
three-body binding equilibrium complicated by potential
cooperativity or avidity effects arising from protein-protein
interactions (Douglass, Miller et al. 2013), all of which precludes
direct interpretation of the binding data. However, assuming
constant concentrations of BRD4.sub.BD1, DDB1.DELTA.B-CRBN, and
fluorescent labels, as well as similar binding conformations, the
peak height of the TR-FRET can be used as an indication for the
amount of tertiary complex formation (containing BRD4.sub.BD1/BD2,
dBET, and CRBN) (Douglass, Miller et al. 2013). The peak height of
TR-FRET dBET dose response data was calculated in GraphPad Prism 7
using Area Under Curve analysis for three independent replicates
(n=3) and the mean peak height and standard deviation
calculated.
[0230] Counter titrations with unlabelled proteins were carried out
by addition of solution of 200 nM
His.sub.6-DDB1.DELTA.B-His.sub.6-CRBN.sub.BODIPY-Spycatcher, 160 nM
biotinylated His.sub.6-Avi-IKZF1.DELTA., 4 nM terbium-coupled
streptavidin and 2 .mu.M of dBET57, incubated for 15 min on ice, to
equal volume of titrated unlabelled His.sub.6-Avi-BRD4.sub.BD1 or
His.sub.6-Avi-BRD4.sub.BD2 to the final assay concentrations.
[0231] The 520/490 nm ratios in IKZF1A TR-FRET assays were plotted
to calculate the half maximal effective concentrations
(EC.sub.50--for unlabelled protein titrations) or IC.sub.50 (for
compound titrations) assuming a single binding site using GraphPad
Prism 7 variable slope equation. The standard deviation in IKZF1A
TR-FRET compound titrations was calculated from three biological
replicates (n=3) as an average of 5 technical replicates per well
per experiment, or as an average of 5 technical replicates of
single experiment for unlabelled protein titrations.
Example 13: Fluorescence Polarization
[0232] Atto565-conjugated lenalidomide (10 nM) was mixed with
increasing concentration of purified
his.sub.6-DDB1.DELTA.B-his.sub.6-CRBN (10 .mu.M final top
concentration, 2-fold, 23 point dilution and DMSO control) in
384-well microplates (Corning, 4514) and incubated for 15 min at
RT. The change in fluorescence polarization was monitored using a
PHERAstar.RTM. FS microplate reader (BMG Labtech) for 20 min in 120
s cycles. The Atto565-lenalidomide bound fraction was calculated as
described (Marks, Qadir et al. 2005) and the K.sub.d was obtained
from a fit in GraphPad Prism 7 from four independent replicates
(n=4).
[0233] Compounds in Atto565-Lenalidomide displacement assay were
dispensed in a 384-well microplate (Corning, 4514) using D300e
Digital Dispenser (HP) normalized to 2% DMSO into 10 nM
Atto565-Leanlidomide, 100 nM DDB1.DELTA.B-CRBN, 50 mM Tris pH 7.5,
100 mM NaCl, 0.1% Pluronic F-68 solution (Sigma), 0.5 mg/ml BSA
(Sigma) containing 2% DMSO (4% DMSO final). Compound titrations
were performed in presence of 0, 1, 5, 20 .mu.M of unbiotinylated
his.sub.6-avi-BRD4.sub.BD1 or his.sub.6-avi-BRD4.sub.BD2 and
incubated for 60 min at RT. The change in fluorescence polarization
was monitored using a PHERAstar.RTM. FS microplate reader (BMG
Labtech) for 20 min in 200 s cycles. Data from two independent
measurements (n=2) was plotted and IC.sub.50 values estimated using
variable slope equation in GraphPad Prism 7.
Example 14: Cellular Degradation Assays
[0234] IKZF1A, BRD2.sub.BD1, BRD2.sub.BD2, BRD3.sub.BD1,
BRD3.sub.BD2, BRD4.sub.BD1, and BRD4.sub.BD2 were subcloned into
mammalian pcDNA5/FRT Vector (Ampicillin and Hygromycin B resistant)
modified to contain MCS-eGFP-P2A-mCherry. Stable cell lines
expressing eGFP-protein fusion and mCherry reporter were generated
using Flip-In 293 system. Plasmid (0.3 .mu.g) and pOG44 (4.7 .mu.g)
DNA were preincubated in 100 .mu.L of Opti-MEM.TM. I (Gibco.RTM.,
Life Technologies) media containing 0.05 mg/ml Lipofectamine.RTM.
2000 (Invitrogen) for 20 min and added to Flip-In 293 cells
containing 1.9 ml of DMEM media (Gibco.RTM., Life Technologies) per
well in a 6-well plate format (Falcon, 353046). Cells were
propagated after 48 h and transferred into a 10 cm.sup.2 plate
(Corning, 430165) in DMEM media containing 50 .mu.g/ml of
Hygromycin B (REF 10687010, Invitrogen) as a selection marker.
Following 2-3 passage cycle FACS (FACSAria II, BD) was used to
enrich for cells expressing eGFP and mCherry.
[0235] Cells were seeded at 30-50% confluency in either 24, 48 or
96 well plates (3524, 3548, 3596 respectively, Costar) a day before
compound treatment. Titrated compounds were incubated with cells
for 5 h following trypsinisation and resuspention in DMEM media,
transferred into 96-well plates (353910, Falcon) and analyzed by
flow cytometer (guava easyCyte.TM. HT, Millipore). Signal from 5000
cells per well was acquired in singlicate or duplicate and the eGFP
and mCherry florescence monitored. Data was analyzed using FlowJo
(FlowJo, LCC). Forward and side scatter outliers, frequently
associated with cell debris, were removed leaving >90% of total
cells, followed by removal of eGFP and mCherry signal outliers,
leaving 88-90% of total cells creating the set used for
quantification. The eGFP protein abundance relative to mCherry was
then quantified as a ten-fold amplified ratio for each individual
cell using the formula: 10.times.eGFP/mCherry. The median of the
ratio was then calculated per set, normalized to the median of the
DMSO ratio, and is denoted as relative abundance. Standard
deviation is calculated from four replicates (n=4) unless described
otherwise.
Example 15: Western Blot for Cellular BRD2/3/4 Degradation
[0236] HEK293T cells were seeded at 90% confluency in 12 well
plates (353043, Falcon), left to attach for 1.5 h, followed by the
compound treatment for 5 h. Primary and secondary antibodies used
included anti-BRD4 at 1:1000 dilution (A301-985A-M, Bethyl
Laboratories), anti-BRD2 at 1:2,000 dilution (A302-582A, Bethyl
Laboratories), anti-BRD3 at 1:500 dilution (ab56342, Abcam.RTM.),
anti-GAPDH at 1:10,000 dilution (G8795, Sigma), IRDye.RTM. 680
Donkey anti-mouse at 1:10,000 dilution (926-68072, LiCor.RTM.) and
IRDye800 Goat anti-rabbit at 1:10,000 dilution (926-32211,
LiCor.RTM.).
Example 16: Sample Preparation and TMT LC-MS3 Mass Spectrometry
Analysis
[0237] MM.1s cell were treated with DMSO, 1 .mu.M dBET23, or dBET70
in biological triplicates for 5 hours and cells harvested by
centrifugation. Lysis buffer (8 M Urea, 1% SDS, 50 mM Tris pH 8.5,
Protease and Phosphatase inhibitors from Roche) was added to the
cell pellets to achieve a cell lysate with a protein concentration
between 2-8 mg mL.sup.-1. A micro-BCA assay (Pierce) was used to
determine the final protein concentration in the cell lysate. 200
.mu.g proteins for each sample were reduced and alkylated as
previously described. Proteins were precipitated using
methanol/chloroform. In brief, four volumes of methanol were added
to the cell lysate, followed by one volume of chloroform, and
finally three volumes of water. The mixture was vortexed and
centrifuged to separate the chloroform phase from the aqueous
phase. The precipitated protein was washed with one volume of
ice-cold methanol. The washed precipitated protein was allowed to
air dry. Precipitated protein was resuspended in 4 M Urea, 50 mM
Tris pH 8.5. Proteins were first digested with LysC (1:50;
enzyme:protein) for 12 hours at 25.degree. C. The LysC digestion
was diluted down in 1 M Urea, 50 mM Tris pH 8.5 and then digested
with trypsin (1:100; enzyme:protein) for another 8 hours at
25.degree. C. Peptides were desalted using a C.sub.18 solid phase
extraction cartridges (Waters). Dried peptides were resuspended in
200 mM EPPS, pH 8.0. Peptide quantification was performed using the
micro-BCA assay (Pierce). The same amount of peptide from each
condition was labelled with tandem mass tag (TMT) reagent (1:4;
peptide:TMT label) (Pierce). The 10-plex labelling reactions were
performed for 2 hours at 25.degree. C. Modification of tyrosine
residue with TMT was reversed by the addition of 5% hydroxyl amine
for 15 minutes at 25.degree. C. The reaction was quenched with 0.5%
TFA and samples were combined at a 1:1:1:1:1:1:1:1:1:1 ratio.
Combined samples were desalted and offline fractionated into 96
fractions using an aeris peptide xb-c18 column (phenomenex) at pH
8.0. Fractions were recombined in a non-continuous manner into 24
fractions and every second fraction was used for subsequent mass
spectrometry analysis.
[0238] Data were collected using an Orbitrap Fusion Lumos mass
spectrometer (Thermo Fisher Scientific, San Jose, Calif., USA)
coupled with a Proxeon EASY-nLCTM 1200 LC pump (Thermo Fisher
Scientific). Peptides were separated on a 75 .mu.m inner diameter
microcapillary column packed with 35 cm of Accucore C18 resin (2.6
.mu.m, 100 .ANG., Thermo Fisher Scientific). Peptides were
separated using a 3 hr gradient of 6-27% acetonitrile in 0.125%
formic acid with a flow rate of 400 nL/min.
[0239] Each analysis used an MS.sup.3-based TMT method as described
previously (McAlister, Nusinow et al. 2014). The data were acquired
using a mass range of m/z 350-1350, resolution 120,000, AGC target
1.times.10.sup.6, maximum injection time 100 ms, dynamic exclusion
of 120 seconds for the peptide measurements in the Orbitrap. Data
dependent MS.sup.2 spectra were acquired in the ion trap with a
normalized collision energy (NCE) set at 35%, AGC target set to
1.8.times.10.sup.4 and a maximum injection time of 120 ms. MS.sup.3
scans were acquired in the Orbitrap with a HCD collision energy set
to 55%, AGC target set to 1.5.times.10.sup.5, maximum injection
time of 150 ms, resolution at 50,000 and with a maximum synchronous
precursor selection (SPS) precursors set to 10.
[0240] Proteome Discoverer.TM. 2.1 (Thermo Fisher) was used to
for.RAW file processing and controlling peptide and protein level
false discovery rates, assembling proteins from peptides, and
protein quantification from peptides. MS/MS spectra were searched
against a Uniprot human database (September 2016) with both the
forward and reverse sequences. Database search criteria are as
follows: tryptic with two missed cleavages, a precursor mass
tolerance of 50 ppm, fragment ion mass tolerance of 1.0 Da, static
alkylation of cysteine (57.02146 Da), static TMT labelling of
lysine residues and N-termini of peptides (229.16293 Da), and
variable oxidation of methionine (15.99491 Da). TMT reporter ion
intensities were measured using a 0.003 Da window around the
theoretical m/z for each reporter ion in the MS.sup.3 scan. Peptide
spectral matches with poor quality MS.sup.3 spectra were excluded
from quantitation (<summed signal-to-noise across 10 channels
and <0.5 precursor isolation specificity).
[0241] Reporter ion intensities were normalised and scaled in the R
framework (Team 2013). Statistical analysis was carried out using
the limma package within the R framework (Ritchie, Phipson et al.
2015).
Example 17: Protein Docking
[0242] All protein docking was carried out using Rosetta 3.7
provided through SBGrid (Morin, Eisenbraun et al. 2013). Input
models were downloaded from the PDB (hsCRBN pdb: 4tz4; BRD4.sub.BD1
pdb: 3mxf, BRD4.sub.BD2 pdb: 2ouo, and hsCSNK1A1 pdb: 5fqd). Ligand
conformers were generated using OpenEye Omega (OpenEye scientific)
and parameter files generated using Rosetta `molfile_to_params.py`.
Relevant PDB's were combined into a single file and prepared for
docking using the Rosetta dockingprepackprotoca program. Initial
global docking was performed using Rosetta dockingprotocol mpi'
with the following command line options:
partners A_B--dock_pert 5 25--randomize2--ex1 ex2aro-nstruct 20000
providing the combined pdb and ligand specific parameter files as
input.
[0243] For Ck1.alpha., and the initial analysis of BRD4.sub.BD1,
the two lowest scoring solutions were used for local perturbation
docking with Rosetta dockingprotocol mpi' with the following
command line options:
partners A_B--dock_pert 8 18--ex1 ex2aro-nstruct 2000
[0244] To assess the landscape of possible binding modes for
BRD4.sub.BD1 and BRD4.sub.BD2, the top 200 lowest scoring docking
decoys were selected and hierarchical clustered according to the
compound centroids and orientations. The lowest scoring model of
each cluster was loaded into pymol and decoys that would position
the thalidomide and JQ1 binding sites on CRBN and BRD4.sub.BD1/2,
respectively, more than 30 .ANG. apart. The remaining decoys were
considered.
[0245] Methods were developed for the design of heterobifunctional
compounds based on computational protein-protein docking, including
methods for analysis of the docking results and the inference of
design information for chemical synthesis. These methods were
applied to the BET family protein BRD4 to synthesize working
examples.
[0246] Protein-protein docking programs such as Rosetta output
docked poses of the two proteins. In one embodiment, BRD4.sub.BD1
was docked with CRBN in the presence of the ligands, JQ1 and
lenalidomide respectively, resulting in 10,000 scored poses. Then,
the shortest distance paths between a set of solvent exposed atoms
on both ligands was calculated and plotted those as a histogram of
the distances (FIG. 18). Histogram of 10,000 distances and the
distances from top 200 scoring poses present clearly distinct
profiles. The profile of all poses approximates a normal
distribution, whereas the profile of the top 200 poses has clear
regions (i.e., clusters) of distances that occurred with higher
frequency (FIG. 18). These clusters indicate a preference for the
complex formation in these particular distance constraints.
[0247] Data analysis and statistics for all steps were performed
using the R framework (Team 2013) or Matlab.
Example 18: Synthesis of dBET6
##STR00019##
[0248]
2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione
[0249] 3-Hydroxyphthalic anhydride (1.641 g, 10 mmol, 1 eq.) and
3-aminopiperidine-2,6-dione hydrochloride (1.646 g, 10 mmol, 1 eq.)
were dissolved in pyridine (40 mL, 0.25 M) and heated to
110.degree. C. After 14 hours, the mixture was cooled to room
temperature and concentrated under reduced pressure. Purification
by column chromatography (ISCO, 24 g silica column, 0-10% MeOH/DCM)
gave the desired product as a tan solid (2.424 g, 8.84 mmol,
88%).
[0250] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 11.08 (s, 2H),
7.65 (dd, J=8.4, 7.2 Hz, 1H), 7.36-7.28 (m, 1H), 7.25 (dd, J=8.4,
0.6 Hz, 1H), 5.07 (dd, J=12.8, 5.4 Hz, 1H), 2.88 (ddd, J=17.3,
14.0, 5.4 Hz, 1H), 2.63-2.50 (m, 2H), 2.08-1.95 (m, 1H).
##STR00020##
tert-Butyl
2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetate
[0251] 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione
(1.568 g, 5.71 mmol, 1 eq.) was dissolved in DMF (57 mL, 0.1 M) at
room temperature. Potassium carbonate (1.19 g, 8.58 mmol, 1.5 eq.)
and tert-butyl bromoacetate (0.843 mL, 5.71 mmol, 1 eq.) were then
added. After 2 hours, the mixture was diluted with EtOAc and washed
once with water, then twice with brine. The organic layer was dried
over sodium sulfate, filtered and concentrated under reduced
pressure. Purification by column chromatography (ISCO, 24 g silica
column, 0-100% EtOAc/hexanes, 21 minute gradient) gave the desired
product as a cream colored solid (2.06 g, 5.30 mmol, 93%).
[0252] .sup.1H NMR (500 MHz, C.sub.DCl3) .delta. 7.94 (s, 1H), 7.67
(dd, J=8.4, 7.3 Hz, 1H), 7.52 (d, J=6.8 Hz, 1H), 7.11 (d, J=8.3 Hz,
1H), 4.97 (dd, J=12.3, 5.3 Hz, 1H), 4.79 (s, 2H), 2.95-2.89 (m,
1H), 2.85-2.71 (m, 2H), 2.14 (dtd, J=10.2, 5.0, 2.7 Hz, 1H), 1.48
(s, 9H).
[0253] LCMS 389.33 (M+H).sup.+.
##STR00021##
2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetic
acid
[0254] tert-Butyl
2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetate
(2.06 g, 5.30 mmol, 1 eq.) was dissolved in trifluoroacetic acid
(TFA) (53 mL, 0.1M) at room temperature. After 4 hours, the
solution was diluted with DCM and concentrated under reduced
pressure. The resultant cream colored solid (1.484 g, 4.47 mmol,
84%) was deemed sufficiently pure and carried onto the next step
without further purification.
[0255] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.11 (s, 1H),
7.79 (dd, J=8.4, 7.4 Hz, 1H), 7.48 (d, J=7.2 Hz, 1H), 7.39 (d,
J=8.6 Hz, 1H), 5.10 (dd, J=12.8, 5.4 Hz, 1H), 4.99 (s, 2H),
2.93-2.89 (m, 1H), 2.63-2.51 (m, 2H), 2.04 (ddd, J=10.5, 5.4, 3.1
Hz, 1H).
[0256] LCMS 333.25 (M+H).sup.+.
##STR00022##
tert-Butyl
(8-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido-
)octyl)carbamate
[0257] Boc-1,8-diaminooctane (2.10 g, 8.59 mmol, 1.1 eq.) was
dissolved in DMF (86 mL). In a separate flask,
2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetic
acid (2.60 g, 7.81 mmol, 1 eq.) was dissolved in DMF (78 mL). The
solution of Boc-1,8-diaminooctane in DMF was then added, followed
by N,N-diisopropylethylamine (DIPEA) (4.08 mL, 23.4 mmol. 3 eq.)
and
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate 0 (2.97 g, 7.81 mmol, 1 eq.). The
mixture was stirred for 19 hours at room temperature, then diluted
with EtOAc (600 mL). The organic layer was washed sequentially with
200 mL of half saturated sodium chloride, 200 mL 10% citric acid
(aq.), 200 mL of half saturated sodium chloride, 200 mL of
saturated sodium bicarbonate (aq.), 200 mL water and twice with 200
mL brine. The organic layer was dried over sodium sulfate, filtered
and concentrated under reduced pressure. Purification by column
chromatography (ISCO, 40 g column, 0-5% MeOH/DCM, 35 minute
gradient) gave the desired product as a white solid (3.53 g, 6.32
mmol, 81%).
[0258] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 8.49 (s, 1H), 7.74
(dd, J=8.3, 7.4 Hz, 1H), 7.55 (d, J=7.2 Hz, 1H), 7.39 (t, J=5.3 Hz,
1H), 7.19 (d, J=8.4 Hz, 1H), 4.97 (dd, J=12.4, 5.3 Hz, 1H), 4.63
(d, J=2.2 Hz, 2H), 4.59 (d, J=10.0 Hz, 1H), 3.36 (q, J=6.9 Hz, 2H),
3.12-3.03 (m, 2H), 2.95-2.72 (m, 3H), 2.16 (ddt, J=10.3, 5.2, 2.7
Hz, 1H), 1.59 (p, J=7.1 Hz, 2H), 1.37 (d, J=67.6 Hz, 19H).
[0259] LCMS 559.47 (M+H).sup.+.
##STR00023##
N-(8-Aminooctyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl-
)oxy)acetamide trifluoroacetate
[0260] tert-Butyl
(8-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamid-
o)octyl)carbamate (3.53 g, 6.32 mmol, 1 eq.) was dissolved in TFA
(63 mL, 0.1M) and heated to 50.degree. C. After 1 hour, the mixture
was cooled to room temperature, diluted with MeOH and concentrated
under reduced pressure. The crude material was triturated with
diethyl ether and dried under vacuum to give a white solid (2.93 g,
5.12 mmol, 81%).
[0261] .sup.1H NMR (500 MHz, MeOD) .delta. 7.82 (dd, J=8.4, 7.4 Hz,
1H), 7.55 (d, J=7.2 Hz, 1H), 7.44 (d, J=8.4 Hz, 1H), 5.14 (dd,
J=12.5, 5.5 Hz, 1H), 4.76 (s, 2H), 3.33 (dd, J=6.8, 1.8 Hz, 1H),
3.30 (s, 1H), 2.94-2.85 (m, 3H), 2.80-2.69 (m, 2H), 2.19-2.11 (m,
1H), 1.60 (dq, J=24.8, 7.0 Hz, 4H), 1.37 (s, 8H).
[0262] LCMS 459.45 (M+H).sup.+.
##STR00024##
[0263] dBET6
[0264]
(S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,24][1,2,4]tri-
azolo[4,3-a][1,4]diazepin-6-yl)acetic acid (JQ-acid) (0.894 g, 2.23
mmol, 1 eq.) and
N-(8-aminooctyl)-2-((2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl)oxy)acetamide trifluoroacetate (1.277 g) were dissolved in DMF
(22.3 mL, 0.1M) at room temperature. DIPEA (1.17 mL, 6.69 mmol, 3
eq.) was added, followed by HATU (0.848 g, 2.23 mmol, 1 eq.). The
mixture was stirred for 23 hours, and then diluted with EtOAc. The
organic layer was washed with saturated sodium bicarbonate, water
and three times with brine. The organic layer was then dried under
sodium sulfate, filtered and concentrated under reduced pressure.
Purification by column chromatography (ISCO, 40 g column, 4-10%
MeOH/DCM, 35 minute gradient) gave dBET6 as a cream colored solid
(1.573 g, 1.87 mmol, 84%).
[0265] .sup.1H NMR (500 MHz, MeOD) .delta. 7.80 (dd, J=8.3, 7.5 Hz,
1H), 7.53 (d, J=7.3 Hz, 1H), 7.46-7.37 (m, 5H), 5.11 (ddd, J=12.6,
8.2, 5.5 Hz, 1H), 4.75 (s, 2H), 4.63 (dd, J=9.0, 5.2 Hz, 1H), 3.41
(ddd, J=14.9, 9.0, 2.2 Hz, 1H), 3.30-3.14 (m, 5H), 2.86 (ddt,
J=19.8, 14.6, 5.2 Hz, 1H), 2.78-2.66 (m, 5H), 2.44 (s, 3H), 2.13
(ddq, J=15.3, 7.7, 4.8, 3.8 Hz, 1H), 1.69 (s, 3H), 1.61-1.51 (m,
4H), 1.35 (s, 8H).
[0266] .sup.13C NMR (126 MHz, MeOD) .delta. 174.49, 172.65, 171.30,
169.80, 168.28, 167.74, 166.18, 157.03, 156.24, 152.18, 138.19,
138.08, 137.97, 134.92, 133.52, 133.23, 132.02, 131.99, 131.33,
129.76, 121.65, 119.30, 117.94, 69.36, 55.27, 50.57, 40.49, 40.13,
38.84, 32.19, 30.49, 30.34, 30.31, 30.22, 27.92, 27.82, 23.64,
14.42, 12.92, 11.60.
[0267] LCMS 841.48 (M+H).sup.+.
Example 19: Synthesis of dBET23
##STR00025##
[0269] dBET23
[0270] A 0.1 M solution of
N-(8-aminooctyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl-
)oxy)acetamide trifluoroacetate in DMF (220 microliters, 0.0220
mmol, 1 eq.) was added to
(S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,-
2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-carboxylic acid (9.87
mg, 0.0220 mmol, 1 eq.) at room temperature. DIPEA (11.5
microliters, 0.0660 mmol, 3 eq.) and HATU (8.4 mg, 0.0220 mmol, 1
eq.) were added. The mixture was then stirred for 21 hours, then
diluted with EtOAc and washed with saturated sodium bicarbonate,
water and brine. The organic layer was dried over sodium sulfate,
filtered and concentrated under reduced pressure. Purification by
column chromatography (ISCO, 4 g silica column, 0-10% MeOH/DCM, 25
minute gradient) gave the desired product as a white solid (8.84
mg, 0.00998 mmol, 45%).
[0271] .sup.1H NMR (400 MHz, MeOD) .delta. 7.81 (dd, J=8.4, 7.4 Hz,
1H), 7.53 (d, J=7.3 Hz, 1H), 7.50-7.39 (m, 5H), 5.12 (dd, J=12.6,
5.4 Hz, 1H), 4.75 (s, 2H), 4.68 (t, J=7.2 Hz, 1H), 3.76 (s, 3H),
3.54 (d, J=7.2 Hz, 2H), 3.39-3.32 (m, 3H), 3.29 (s, 1H), 2.90-2.83
(m, 1H), 2.79-2.68 (m, 5H), 2.14 (dd, J=8.9, 3.7 Hz, 1H), 1.99 (s,
3H), 1.65-1.53 (m, 4H), 1.36 (d, J=6.5 Hz, 8H).
[0272] LCMS 885.47 (M+H).sup.+.
Example 20: Synthesis of dBET55
##STR00026##
[0273] tert-Butyl
(1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2-oxo-6,9,-
12,15,18,21,24,27,30-nonaoxa-3-azadotriacontan-32-yl)carbamate
[0274] tert-Butyl
(29-amino-3,6,9,12,15,18,21,24,27-nonaoxanonacosyl) carbamate
(422.53 mg, 0.759 mmol, 1 eq.) as a solution in 15.18 ml DMF (0.1
M) was added to
2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)
acetic acid (252.26 mg, 0.759, 1 eq.). DIPEA (376.45 2.277 mmol, 3
eq.) was added, followed by HATU (288.6 mg, 0.759 mmol, 1 eq.). The
mixture was stirred for 17 hours at room temperature. The mixture
was then diluted with EtOAc and washed with saturated sodium
bicarbonate, water then brine. The organic layer was dried over
sodium sulfate, filtered and condensed to give a white solid (255.8
mg, 39% yield). The crude material was purified by column
chromatography (ISCO, 12 g silica column, 0 to 10% MeOH/DCM 25
minute gradient) to give a white solid (105.3 mg, 16% yield).
[0275] .sup.1H NMR (500 MHz, MeOD) .delta. 7.80 (dd, J=8.4, 7.3 Hz,
1H), 7.50 (dd, J=7.3 Hz, 1H), 7.43 (dd, J=8.5 Hz, 1H), 5.12 (dd,
J=12.8, 5.5 Hz, 1H), 3.61 (m, J=8.2, 5.6, 2.6 Hz, 36H), 3.50 (dd,
J=5.6, 1.9 Hz, 4H), 3.22 (q, J=5.5 Hz, 2H), 2.90 (ddd, J=17.5,
13.9, 5.3 Hz, 1H), 2.80-2.70 (m, 2H), 2.17 (m, J=13.1, 5.8, 2.8 Hz,
1H), 1.43 (s, 9H).
[0276] LCMS 871.35 (M+H).sup.+.
##STR00027##
N-(29-Amino-3,6,9,12,15,18,21,24,27-nonaoxanonacosyl)-2-((2-(2,6-dioxopip-
eridin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide
trifluoroacetate salt
[0277] tert-Butyl
(1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2-oxo-6,9,-
12,15,18,21,24,27,30-nonaoxa-3-azadotriacontan-32-yl)carbamate
(105.3 mg, 0.121 mmol, 1 eq.) was added to 1.21 ml TFA (0.1M) and
was stirred for 2 hours at 50.degree. C. The mixture was diluted
with methanol and condensed to give a white solid (104.28, 97%
yield) with no further purification.
[0278] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.11 (s, 1H),
8.00 (s, J=5.8 Hz, 1H), 7.82 (dd, J=7.9 Hz, 1H), 7.75-7.71 (s, 2H),
7.50 (dd, J=7.3 Hz, 1H), 7.40 (dd, J=8.6 Hz, 1H), 5.11 (dd, J=12.8,
5.4 Hz, 1H), 4.79 (s, 2H), 3.91-3.41 (m, 36H), 3.32 (t, J=5.7 Hz,
2H), 2.98 (m, J=5.5 Hz, 2H), 2.90 (ddd, J=18.1, 14.0, 5.3 Hz, 1H),
2.63-2.54 (m, 2H), 2.05 (dd, J=12.3, 6.1 Hz, 1H).
[0279] LCMS 771.80 (M+H).sup.+.
##STR00028##
[0280] dBET55
[0281] A 0.1 M solution of
N-(29-amino-3,6,9,12,15,18,21,24,27-nonaoxanonacosyl)-2-((2-(2,6-dioxopip-
eridin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide
trifluoroacetate in DMF (200 microliters, 0.020 mmol, 1 eq.) was
added to JQ-acid (8.0 mg, 0.020 mmol, 1 eq.) at room temperature.
DIPEA (10.5 microliters, 0.060 mmol, 3 eq.) and HATU (7.6 mg, 0.020
mmol, 1 eq.) were added. After 18 hours the mixture was diluted
with EtOAc and washed with saturated sodium bicarbonate, water and
brine. The combined organic layer was dried over sodium sulfate,
filtered and concentrated under reduced pressure. Purification by
column chromatography (ISCO, 4 g silica column, 0-10% MeOH/DCM, 25
minute gradient) gave the desired product (10.55 mg, 0.00914 mmol,
46%).
[0282] .sup.1H NMR (500 MHz, MeOD) .delta. 7.82 (dd, J=8.4, 7.4 Hz,
1H), 7.55 (d, J=7.0 Hz, 1H), 7.49-7.41 (m, 5H), 5.13 (dd, J=12.6,
5.5 Hz, 1H), 4.80 (s, 2H), 4.65 (dd, J=9.1, 5.1 Hz, 1H), 3.68-3.58
(m, 36H), 3.53-3.44 (m, 5H), 2.94-2.86 (m, 1H), 2.81-2.70 (m, 5H),
2.46 (s, 3H), 2.19-2.13 (m, 1H), 1.74-1.69 (m, 3H).
[0283] LCMS 1153.59 (M+H).sup.+.
Example 21: Synthesis of dBET57
##STR00029##
[0284]
2-(2,6-Dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione
[0285] 3-Fluorophthalic anhydride (1.66 g, 10 mmol, 1 eq.) and
3-aminopiperidine-2,6-dione hydrochloride salt (1.81 g, 11 mmol,
1.1 eq.) were dissolved in AcOH (25 mL) followed by potassium
acetate (3.04 g, 31 mmol, 3.1 eq.). The mixture was fitted with an
air condenser and heated to 90.degree. C. After 16 hours, the
mixture was diluted with 100 mL water and cooled over ice. The
slurry was then centrifuged (4000 rpm, 20 minutes, 4.degree. C.)
and decanted. The remaining solid was then resuspended in water,
centrifuged and decanted again. The solid was then dissolved in
MeOH and filtered through a silica plug (that had been pre-wetted
with MeOH), washed with 50% MeOH/DCM and concentrated under reduced
pressure to yield the desired product as a grey solid (2.12 g, 7.68
mmol, 77%).
[0286] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.13 (s, 1H),
7.98-7.91 (m, 1H), 7.79 (d, J=7.3 Hz, 1H), 7.74 (t, J=8.8 Hz, 1H),
5.16 (dd, J=12.9, 5.4 Hz, 1H), 2.89 (ddd, J=17.2, 14.0, 5.5 Hz,
1H), 2.61 (ddd, J=17.1, 4.4, 2.4 Hz, 1H), 2.57-2.50 (m, 1H), 2.06
(dtd, J=13.0, 5.4, 2.3 Hz, 1H).
[0287] LCMS 277.21 (M+H).sup.+.
##STR00030##
tert-Butyl
(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)
carbamate
[0288] A stirred solution of
2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (174 mg,
0.630 mmol, 1 eq.) in DMF (6.3 mL, 0.1 M) was added DIPEA (220
.mu.L, 1.26 mmol, 2 eq.) and 1-Boc-ethylendiamine (110 .mu.L, 0.693
mmol, 1.1 eq.). The reaction mixture was heated to 90.degree. C.
overnight, whereupon it was cooled to room temperature and taken up
in EtOAc (30 mL) and water (30 mL). The organic layer was washed
with brine (3.times.20 mL), dried over Na.sub.2SO.sub.4 and
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (0-10% MeOH in DCM) to give the title
compound as a yellow solid (205 mg, 79%).
[0289] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 8.08 (bs, 1H),
7.50 (dd, J=8.5, 7.1 Hz, 1H), 7.12 (d, J=7.1 Hz, 1H), 6.98 (d,
J=8.5 Hz, 1H), 6.39 (t, J=6.1 Hz, 1H), 4.96-4.87 (m, 1H), 4.83 (bs,
1H), 3.50-3.41 (m, 2H), 3.41-3.35 (m, 2H), 2.92-2.66 (m, 3H),
2.16-2.09 (m, 1H), 1.45 (s, 9H).
[0290] LCMS 417.58 (M+H).sup.+.
##STR00031##
4-((2-Aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
trifluoroacetate salt
[0291] To a stirred solution of tert-butyl
(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)car-
bamate (205 mg, 0.492 mmol, 1 eq.) in dichloromethane (2.25 mL) was
added trifluoroacetic acid (0.250 mL). The reaction mixture was
stirred at room temperature for 4 h, whereupon the volatiles were
removed in vacuo. The title compound was obtained as a yellow solid
(226 mg, >95%), that was used without further purification.
[0292] .sup.1H NMR (500 MHz, MeOD) .delta. 7.64 (d, J=1.4 Hz, 1H),
7.27-7.05 (m, 2H), 5.10 (dd, J=12.5, 5.5 Hz, 1H), 3.70 (t, J=6.0
Hz, 2H), 3.50-3.42 (m, 2H), 3.22 (t, J=6.0 Hz, 1H), 2.93-2.85 (m,
1H), 2.80-2.69 (m, 2H), 2.17-2.10 (m, 1H).
[0293] LCMS 317.53 (M+H).sup.+.
##STR00032##
[0294] dBET57
[0295] JQ-acid (8.0 mg, 0.0200 mmol, 1 eq.) and
2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethan-1-am-
inium 2,2,2-trifluoroacetate (8.6 mg, 0.0200 mmol, 1 eq.) were
dissolved in DMF (0.200 mL, 0.1 M) at room temperature. DIPEA (17.4
.mu.L, 0.100 mmol, 5 eq.) and HATU (7.59 mg, 0.0200 mmol, 1 eq.)
were then added and the mixture was stirred at room temperature
overnight. The reaction mixture was taken up in EtOAc (15 mL), and
washed with saturated (satd.) aqueous NaHCO.sub.3 (aq.) (15 mL),
water (15 mL) and brine (3.times.15 mL). The organic layer was
dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue
was purified by flash column chromatography on silica gel (0-10%
MeOH in DCM, Rf=0.3 (10% MeOH in DCM)) to give the title compound
as a bright yellow solid (11.2 mg, 80%).
[0296] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.49 (bs, 0.6H),
8.39 (bs, 0.4H), 7.51-7.43 (m, 1H), 7.38 (d, J=7.8 Hz, 2H), 7.29
(dd, J=8.8, 1.7 Hz, 2H), 7.07 (dd, J=7.1, 4.9 Hz, 1H), 6.97 (dd,
J=8.6, 4.9 Hz, 1H), 6.48 (t, J=5.9 Hz, 1H), 6.40 (t, J=5.8 Hz,
0.6H), 4.91-4.82 (m, 0.4H), 4.65-4.60 (m, 1H), 3.62-3.38 (m, 6H),
2.87-2.64 (m, 3H), 2.63 (s, 3H), 2.40 (s, 6H), 2.12-2.04 (m, 1H),
1.67 (s, 3H), rotamers;
[0297] LCMS 700.34 (M+H).sup.+.
Example 22: Synthesis of dBET70
##STR00033##
[0298] tert-Butyl
(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octyl)
carbamate
[0299] 2-(2,6-Dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione
(552.4 mg, 2.0 mmol, 1 eq.) and tert-butyl (8-aminooctyl)carbamate
(537.6 mg, 2.2 mmol, 1.1 eq.) were dissolved in N-methylpyrrolidone
(NMP) (10 mL). DIPEA (697 microliters, 4.0 mmol, 2 eq.) was added
and the mixture was heated to 90.degree. C. After 21 hours the
mixture was cooled to room temperature and diluted with EtOAc. The
organic layer was washed with 10% citric acid (aq), brine,
saturated sodium bicarbonate (aq.), water, and three times with
brine. The organic layer was then dried over sodium sulfate,
filtered and concentrated under reduced pressure. The material was
purified by column chromatography (ISCO, 12 g column, 0-5%
MeOH/DCM, 25 minute gradient) to give the desired product as a
yellow solid (0.62 g, 1.24 mmol, 62%).
[0300] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 8.51 (s, 1H),
7.49-7.44 (m, 1H), 7.06 (d, J=7.1 Hz, 1H), 6.86 (d, J=8.6 Hz, 1H),
6.22 (t, J=5.4 Hz, 1H), 4.91 (dd, J=12.2, 5.3 Hz, 1H), 4.56 (s,
1H), 3.24 (q, J=6.7 Hz, 2H), 3.07 (t, J=12.7 Hz, 2H), 2.89-2.67 (m,
3H), 2.11 (dq, J=10.3, 3.6, 2.7 Hz, 1H), 1.64 (p, J=7.0 Hz, 2H),
1.36 (d, J=61.0 Hz, 19H).
[0301] .sup.13C NMR (126 MHz, CDCl.sub.3) .delta. 171.47, 169.60,
168.68, 167.73, 156.06, 147.06, 136.15, 132.57, 116.71, 111.39,
109.91, 79.11, 48.95, 42.68, 40.66, 31.49, 30.09, 29.25, 29.20,
28.51, 26.89, 26.75, 22.89.
[0302] LCMS 501.39 (M+H).sup.+.
##STR00034##
4-((8-Aminooctyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
trifluoroacetate salt
[0303] tert-Butyl
(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octyl)
carbamate (0.55 g, 1.099 mmol, 1 eq.) was dissolved in TFA (11 mL)
and heated to 50.degree. C. After 40 minutes, the mixture was
concentrated under reduced pressure, triturated with Et.sub.2O, and
dried under high vacuum to yield a yellow residue (523 mg, 1.016
mmol, 93%) that was used without further purification.
[0304] .sup.1H NMR (500 MHz, MeOD) .delta. 7.59-7.51 (m, 1H), 7.04
(dd, J=7.9, 1.7 Hz, 2H), 5.06 (dd, J=12.4, 5.5 Hz, 1H), 3.34 (d,
J=7.0 Hz, 2H), 2.95-2.81 (m, 3H), 2.79-2.66 (m, 2H), 2.15-2.08 (m,
1H), 1.67 (tt, J=12.2, 7.2 Hz, 4H), 1.43 (d, J=22.2 Hz, 8H).
[0305] LCMS 401.39 (M+H).sup.+.
##STR00035##
[0306] dBET70
[0307]
(S)-4-(4-Chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thi-
eno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-carboxylic acid
(201 mg, 0.452 mmol, 1 eq.) and
4-((8-aminooctyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
trifluoroacetate salt (232.5 mg, 0.452 mmol, 1 eq.) were dissolved
in DMF (4.5 mL). DIPEA (236 .mu.L, 1.355 mmol, 3 eq.) and HATU
(171.9 mg, 0.452 mmol, 1 eq.) were added and the mixture was
stirred for 18 hours at room temperature. The mixture was then
diluted with EtOAc, and washed three times with 1M HCl (aq), then
once with brine, saturated sodium bicarbonate, water and brine. The
organic layer was then dried over sodium sulfate, filtered and
concentrated under reduced pressure. Purification by column
chromatography (ISCO, 24 g silica column, 0-6% MeOH/DCM, 35 minute
gradient) to give the desired product as a yellow solid (224.92 mg,
0.2719 mmol, 60%).
[0308] .sup.1H NMR (500 MHz, MeOD) .delta. 7.54-7.50 (m, 1H), 7.45
(d, J=8.5 Hz, 2H), 7.42-7.38 (m, 2H), 7.00 (dd, J=7.8, 2.9 Hz, 2H),
5.00 (ddd, J=12.8, 5.4, 3.1 Hz, 1H), 4.66 (t, J=7.1 Hz, 1H), 3.75
(s, 3H), 3.53 (d, J=7.3 Hz, 2H), 3.37 (dq, J=15.7, 8.3, 7.7 Hz,
2H), 3.29 (d, J=6.9 Hz, 2H), 2.85 (ddd, J=18.3, 13.9, 5.1 Hz, 1H),
2.77-2.64 (m, 5H), 2.11-2.05 (m, 1H), 1.97 (s, 3H), 1.64 (dq,
J=20.8, 6.8 Hz, 4H), 1.41 (d, J=21.1 Hz, 8H).
[0309] .sup.13C NMR (126 MHz, MeOD) .delta. 174.60, 173.08, 171.59,
170.79, 169.25, 165.54, 163.65, 156.83, 152.44, 148.25, 138.20,
137.94, 137.86, 137.81, 137.22, 133.86, 132.42, 131.84, 131.26,
129.89, 117.96, 111.73, 110.94, 54.89, 52.47, 50.16, 43.37, 41.25,
37.13, 32.21, 30.29, 30.22, 30.17, 27.87, 27.78, 23.79, 16.57,
11.68.
[0310] LCMS 827.60 (M+H).sup.+.
Example 23: Synthesis of dBET72
##STR00036##
[0311]
2-(2,6-Dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione
[0312] 4-Fluorophthalic anhydride (3.32 g, 20 mmol, 1 eq.) and
3-aminopiperidine-2,6-dione hydrochloride salt (3.620 g, 22 mmol,
1.1 eq.) were dissolved in AcOH (50 mL) followed by potassium
acetate (6.08 g, 62 mmol, 3.1 eq.). The mixture was fitted with an
air condenser and heated to 90.degree. C. After 16 hours, the
mixture was diluted with 200 mL water and cooled over ice. The
slurry was then centrifuged (4000 rpm, 20 minutes, 4.degree. C.)
and decanted. The remaining solid was then resuspended in water,
centrifuged and decanted again. The solid was then dissolved in
MeOH and filtered through a silica plug (that had been pre-wetted
with MeOH), washed with 50% MeOH/DCM and concentrated under reduced
pressure to yield the desired product as a grey solid (2.1883 g,
7.92 mmol, 40%).
[0313] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.13 (s, 1H),
8.01 (dd, J=8.3, 4.5 Hz, 1H), 7.85 (dd, J=7.4, 2.2 Hz, 1H), 7.72
(ddd, J=9.4, 8.4, 2.3 Hz, 1H), 5.16 (dd, J=12.9, 5.4 Hz, 1H), 2.89
(ddd, J=17.2, 13.9, 5.5 Hz, 1H), 2.65-2.51 (m, 2H), 2.07 (dtd,
J=12.9, 5.3, 2.2 Hz, 1H).
[0314] LCMS 277.22 (M+H).sup.+.
##STR00037##
tert-Butyl
(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)octyl)car-
bamate
[0315] 2-(2,6-Dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione
(294 mg, 1.06 mmol, 1 eq.) and tert-butyl (8-aminooctyl)carbamate
(286 mg, 1.17 mmol, 1.1 eq.) were dissolved in NMP (5.3 mL). DIPEA
(369 microliters, 2.12 mmol, 2 eq.) was added and the mixture was
heated to 90.degree. C. After 19 hours the mixture was cooled to
room temperature and diluted with EtOAc. The organic layer was
washed with water and three times with brine. The organic layer was
then dried over sodium sulfate, filtered and concentrated under
reduced pressure. The material was purified by column
chromatography (ISCO, 12 g column, 0-10% MeOH/DCM, 30 minute
gradient) to give the desired product as a brown solid (0.3345 g,
0.6682 mmol, 63%).
[0316] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 8.12 (s, 1H), 7.62
(d, J=8.3 Hz, 1H), 7.02 (s, 1H), 6.81 (d, J=7.2 Hz, 1H), 4.93 (dd,
J=12.3, 5.3 Hz, 1H), 4.51 (s, 1H), 3.21 (t, J=7.2 Hz, 2H), 3.09 (d,
J=6.4 Hz, 2H), 2.90 (dd, J=18.3, 15.3 Hz, 1H), 2.82-2.68 (m, 2H),
2.16-2.08 (m, 1H), 1.66 (p, J=7.2 Hz, 2H), 1.37 (d, J=62.3 Hz,
20H).
[0317] LCMS 501.41 (M+H).sup.+.
##STR00038##
5-((8-Aminooctyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
trifluoroacetate salt
[0318] tert-Butyl
(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)octyl)
carbamate (334.5 mg, 0.668 mmol, 1 eq.) was dissolved in TFA (6.7
mL) and heated to 50.degree. C. After 50 minutes, the mixture was
cooled to room temperature, diluted with DCM and concentrated under
reduced pressure, triturated with Et.sub.2O, and dried under high
vacuum to yield a dark yellow foam (253 mg, 0.492 mmol, 74%) that
was used without further purification.
[0319] .sup.1H NMR (500 MHz, MeOD) .delta. 7.56 (d, J=8.4 Hz, 1H),
6.97 (d, J=2.1 Hz, 1H), 6.83 (dd, J=8.4, 2.2 Hz, 1H), 5.04 (dd,
J=12.6, 5.5 Hz, 1H), 3.22 (t, J=7.1 Hz, 2H), 2.94-2.88 (m, 2H),
2.85-2.68 (m, 3H), 2.09 (ddd, J=10.4, 5.4, 3.0 Hz, 1H), 1.70-1.61
(m, 4H), 1.43 (d, J=19.0 Hz, 8H).
[0320] LCMS 401.36 (M+H).sup.+.
##STR00039##
[0321] dBET72
[0322]
5-((8-aminooctyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3--
dione trifluoroacetate salt (10.3 mg, 0.020 mmol, 1 eq.) in DMF
(200 microliters) was added to
(S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,-
2-f][1,2,4]triazolo [4,3-a][1,4]diazepine-2-carboxylic acid (8.9
mg, 0.020 mmol, 1 eq.) at room temperature. DIPEA (10.5
microliters, 0.060 mmol, 3 eq.) was added, followed by HATU (7.6
mg, 0.020 mmol, 1 eq.). After 27 hours, the mixture was diluted
with EtOAc then washed with saturated sodium bicarbonate, water and
brine. The organic layer was dried over sodium sulfate, filtered
and concentrated under reduced pressure. The mixture was purified
by column chromatography (ISCO, 4 g column, 0-10% MeOH/DCM, 25
minute gradient) to give the desired product as a yellow solid
(4.98 mg, 0.00602 mmol, 30%).
[0323] .sup.1H NMR (500 MHz, MeOD) .delta. 7.54 (d, J=8.4 Hz, 1H),
7.49-7.40 (m, 4H), 6.96 (d, J=2.1 Hz, 1H), 6.82 (dd, J=8.4, 2.1 Hz,
1H), 5.02 (dd, J=12.7, 5.5 Hz, 1H), 4.67 (t, J=7.1 Hz, 1H), 3.76
(s, 3H), 3.54 (d, J=7.2 Hz, 2H), 3.41-3.33 (m, 2H), 3.20 (t, J=7.0
Hz, 2H), 2.85 (ddd, J=19.2, 14.0, 5.3 Hz, 1H), 2.77-2.65 (m, 5H),
2.11-2.04 (m, 1H), 1.99 (s, 3H), 1.64 (dt, J=19.3, 7.1 Hz, 4H),
1.43 (d, J=21.8 Hz, 8H).
[0324] LCMS 827.46 (M+H).sup.+.
Example 24: Synthesis of
(S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,-
2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-carboxylic acid
##STR00040## ##STR00041##
[0326] Compound i-1
[0327] (S)-JQ1 (4.57 g, 10 mmol) was dissolved in MeOH (0.25 M).
conc.H.sub.2SO.sub.4 (50 drops) was added to the solution. The
mixture was refluxed overnight. The mixture was concentrated in
vacuo, poured into water, extracted with AcOEt, and washed with
brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered,
and concentrated in vacuo. The residue was purified by flash column
chromatography (AcOEt/MeOH) to give title compound 3.93 g
(95%).
[0328] Compound i-2
[0329] To a mixture of acetic acid (52 mL) and acetic anhydride (30
mL) was added dropwise concentrated sulfuric acid (8 mL). Compound
i-1 (6.04 g, 14.6 mmol) was added, and manganese acetate
(III)*dihydrate (8 g, 29.4 mmol) was further added. The mixture was
stirred at room temperature for 3 days. The reaction mixture was
poured into ice water, and extracted twice with ethyl acetate (300
mL). The organic layer was washed twice with saturated brine (300
mL). The residue was dried over Na.sub.2SO.sub.4, and the solvent
was evaporated to give an oil (5 g), which was used without further
purification.
[0330] Compound i-3
[0331] Compound i-2 (6.0 g, 12.7 mmol) and K.sub.2CO.sub.3 (1.2
eq.) were suspended in MeOH (0.1 M). The mixture was stirred at
room temperature for 2 hours. The mixture was neutralized with IN
HCl, then concentrated in vacuo. The residue was poured into water,
and extracted with DCM. The organic layer was dried over
Na.sub.2SO.sub.4, filtrated, and concentrated in vacuo. The residue
was purified with flash column chromatography (AcOEt/MeOH) to give
title compound 2 g (32% over 2 steps).
[0332] Compound i-4
[0333] Compound i-3 (867 mg, 2.01 mmol) was dissolved in DCM (20
mL). Dess-Martin periodinane (1.2 eq.) was added to the solution at
0.degree. C. The mixture was stirred at room temperature for 2
hours. The mixture was diluted with DCM, washed with saturated
NaHCO.sub.3 solution, dried over Na.sub.2SO.sub.4, and concentrated
in vacuo to give an oil (850 mg). The crude product was used
directly without further purification.
[0334] Compound i-5
[0335] Compound i-4 (850 mg, 1.98 mmoL) was suspended in CH.sub.3CN
(8 mL). Sodium phosphate monobasic (0.97 eq.) in H.sub.2O (3 mL)
solution was added to the suspension. Hydrogen peroxide (5 eq.) was
added to the solution dropwise. Sodium chlorite (1.4 eq.) in
H.sub.2O (2 mL) solution was added to the suspension. The mixture
was stirred for 3 hours. The mixture was diluted with EtOAc,
quenched with Na.sub.2S.sub.2O.sub.3 aq, then, acidified with IN
HCl (pH<4). The mixture was extracted with EtOAc, washed with
brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered,
and concentrated in vacuo. The residue was purified by prep-HPLC to
give compound i-5 (667 mg, 75%).
[0336] .sup.1H NMR (400 MHz, Methanol-d.sub.4) .delta. 7.44 (q,
J=8.8 Hz, 4H), 4.68 (t, J=7.2 Hz, 1H), 3.76 (s, 3H), 3.54 (d, J=7.2
Hz, 2H), 2.74 (s, 3H), 2.09 (s, 3H).
Example 25: Synthesis of ZXH-2-42
##STR00042##
[0337] Dimethyl
3-(2-((tert-butoxycarbonyl)amino)ethoxy)phthalate
[0338] tert-butyl (2-bromoethyl)carbamate (280 mg, 1.25 mmol, 1
eq.) and dimethyl 3-hydroxyphthalate (263 mg, 1.25 mmol, 1 eq.)
were dissolved in DMF (6.25 mL, 0.2 M) followed by potassium
carbonate (345 mg, 2.5 mmol, 2 eq.). The mixture was stirred at
50.degree. C. After the reaction completed, the mixture was cooled
down to room temperature, diluted with EtOAc and washed with water
and brine. The organic layer was dried over sodium sulfate,
filtered and concentrated under reduced pressure. Purification by
column chromatography (ISCO, 12 g silica column, 0-40%
EtOAc/hexane) gave the desired product as a white solid (268 mg,
61%).
[0339] LCMS 354 (M+H).sup.+.
##STR00043##
tert-Butyl
(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)ethyl)
carbamate
[0340] Dimethyl 3-(2-((tert-butoxycarbonyl)amino)ethoxy)phthalate
(268 mg, 0.76 mmol, 1 eq.) was dissolved in EtOH (3.8 mL, 0.2 M)
followed by aqueous 3M NaOH (760 .mu.L, 2.28 mmol, 3 eq.). The
mixture was heated to 80.degree. C. for 4 hours. The mixture was
then cooled down to room temperature, diluted with DCM (14 mL) and
0.5M HCl (5.5 mL). The organic layer was washed with 7 mL of water.
The aqueous layers were combined and extracted three times with 14
mL of chloroform. The combined organic layers were dried over
sodium sulfate, filtered and concentrated to give the material that
was used in the next step.
[0341] LCMS 326 (M+H).sup.+.
[0342] The resultant material and 3-aminopiperidine-2,6-dione
hydrochloride (125 mg, 0.76 mmol, 1 eq.) were dissolved in pyridine
(3.8 mL, 0.2 M) and heated to 110.degree. C. overnight. Then the
mixture was cooled to room temperature and concentrated under
reduced pressure, purified by column chromatography (ISCO, 12 g
silica column, 0-6% MeOH/DCM) to give the desired product (152 mg,
48% for two steps).
[0343] LCMS 417 (M+H).sup.+.
##STR00044##
4-(2-Aminoethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
[0344] tert-Butyl
(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)ethyl)
carbamate (152 mg, 0.37 mmol) was dissolved in TFA (3.7 mL, 0.1 M)
and heated to 50.degree. C. for 3 hours. The mixture was cooled to
room temperature, diluted with methanol and concentrated under
reduced pressure. The material was purified by column
chromatography (ISCO, 4 g silica column, 0-20% 1.75N
NH.sub.3.MeOH/DCM) to give the free base product (101 mg, 86%).
[0345] LCMS 317 (M+H).sup.+.
##STR00045##
[0346] ZXH-2-42
[0347] To a solution of
(S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f]
[1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid (24 mg, 0.06
mmol, 1 eq.) and
4-(2-aminoethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dio-
ne (19 mg, 0.06 mmol, 1 eq.) in DMF (1 ml) were added DIEA (30
.mu.L, 0.18 mmol, 3 eq.) and HATU (27 mg, 0.072 mmol, 1.2 eq.). The
mixture was stirred at room temperature overnight and then purified
by HPLC to give the product as TFA salt (3.8 mg, 8%).
[0348] LCMS 700 (M+H).sup.+.
[0349] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.10 (s, 1H),
8.51 (s, 1H), 7.83 (t, J=7.8 Hz, 1H), 7.59 (d, J=8.6 Hz, 1H),
7.51-7.48 (m, 1H), 7.45-7.39 (m, 4H), 5.09 (s, 1H), 4.57-4.49 (m,
1H), 4.30 (t, J=5.8 Hz, 2H), 3.35 (s, 3H), 3.18-3.13 (m, 2H), 2.86
(s, 1H), 2.60 (s, 3H), 2.42 (s, 2H), 1.62 (s, 3H), 1.27 (s,
2H).
Example 26: Synthesis of ZXH-2-43
##STR00046##
[0350] Dimethyl
3-((8-((tert-butoxycarbonyl)amino)octyl)oxy)phthalate
[0351] tert-Butyl (8-bromooctyl)carbamate (308 mg, 1 mmol, 1 eq.)
and dimethyl 3-hydroxyphthalate (210 mg, 1 mmol, 1 eq.) were
dissolved in DMF (5 mL, 0.2 M) followed by potassium carbonate (276
mg, 2 mmol, 2 eq.). The mixture was stirred at 50.degree. C. After
the reaction reached completion, the mixture was allowed to cool
down to room temperature, diluted with EtOAc and washed with water
and brine. The organic layer was dried over sodium sulfate,
filtered and concentrated under reduced pressure. Purification by
column chromatography (ISCO, 12 g silica column, 0-25%
EtOAc/hexane) gave the desired product as a white solid (315 mg,
72%).
[0352] LCMS 438 (M+H).sup.+.
##STR00047##
tert-Butyl
(8-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)octyl)
carbamate
[0353] Dimethyl
3-((8-((tert-butoxycarbonyl)amino)octyl)oxy)phthalate (315 mg, 0.72
mmol, 1 eq.) was dissolved in EtOH (3.6 mL, 0.2 M) followed by
aqueous 3M NaOH (720 .mu.L, 2.16 mmol, 3 eq.), then the mixture was
heated to 80.degree. C. for 4 hours. The mixture was then cooled
down to room temperature, diluted with DCM (13 mL) and 0.5M HCl
(0.5 mL). The layers were separated and the organic layer was
washed with water (6.5 mL). The aqueous layers were combined and
extracted three times with chloroform (13 ml). The combined organic
layers were dried over sodium sulfate, filtered and condensed to
give the material that was directly used in next step.
[0354] LCMS 410 (M+H).sup.+.
[0355] The resultant material and 3-aminopiperidine-2,6-dione
hydrochloride (118 mg, 0.72 mmol, 1 eq.) were dissolved in pyridine
(3.6 mL, 0.2 M) and heated to 110.degree. C. overnight. Then the
mixture was cooled to room temperature and concentrated under
reduced pressure, purified by column chromatography (ISCO, 12 g
silica column, 0-5% MeOH/DCM) to give the desired product (217 mg,
54% for two steps).
[0356] LCMS 502 (M+H).sup.+.
##STR00048##
4-((8-Aminooctyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
[0357] tert-butyl
(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)octyl)
carbamate (217 mg, 0.43 mmol) was dissolved in TFA (4.3 mL, 0.1 M)
and heated to 50.degree. C. for 3 hours. The mixture was cooled to
room temperature, diluted with MeOH and concentrated under reduced
pressure. The material was purified by column chromatography (ISCO,
4 g silica column, 0-20% 1.75N NH.sub.3.MeOH/DCM) to give the free
base product (152 mg, 88%).
[0358] LCMS 402 (M+H).sup.+.
##STR00049##
[0359] ZXH-2-43
[0360] To a solution of
(S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,-
2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-carboxylic acid (20 mg,
0.045 mmol, 1 eq.) and
4-((8-aminooctyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
(19 mg, 0.045 mmol, 1 eq.) in DMF (1 mL) were added DIEA (23 .mu.L,
0.14 mmol, 3 eq.) and HATU (21 mg, 0.05 mmol, 1.2 eq.). The mixture
was stirred at room temperature overnight and then purified by
HPLC, then by column chromatography (ISCO, 4 g silica column, 0-8%
1.75 N NH.sub.3 in Methanol/DCM) to give the free base product
(22.1 mg, 59%).
[0361] LCMS 828 (M+H).sup.+.
[0362] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.10 (s, 1H),
8.30 (t, J=5.7 Hz, 1H), 7.80 (dd, J=8.5, 7.2 Hz, 1H), 7.51 (d,
J=2.6 Hz, 1H), 7.50 (d, J=2.7 Hz, 2H), 7.48-7.43 (m, 3H), 5.08 (dd,
J=12.8, 5.5 Hz, 1H), 4.58 (dd, J=7.7, 6.6 Hz, 1H), 4.20 (t, J=6.4
Hz, 2H), 3.68 (s, 3H), 3.47 (qd, J=16.6, 7.2 Hz, 2H), 3.34 (s, 1H),
3.29-3.20 (m, 2H), 2.89 (ddd, J=16.9, 13.9, 5.4 Hz, 1H), 2.65 (s,
3H), 2.06-1.99 (m, 1H), 1.91 (s, 3H), 1.76 (p, J=6.6 Hz, 2H), 1.50
(dt, J=33.3, 7.3 Hz, 4H), 1.33 (s, 6H).
Example 27: Synthesis of ZXH-2-45
##STR00050##
[0363] Dimethyl
4-((8-((tert-butoxycarbonyl)amino)octyl)oxy)phthalate
[0364] tert-Butyl (8-bromoethyl)carbamate (182 mg, 0.87 mmol, 1
eq.) and dimethyl 4-hydroxyphthalate (267 mg, 0.87 mmol, 1 eq.)
were dissolved in DMF (4.4 mL) followed by potassium carbonate (239
mg, 1.73 mmol, 2 eq.). The mixture was stirred at 50.degree. C.
After the reaction reached completion, the mixture was allowed to
cool to room temperature, diluted with EtOAc and washed with water
and brine. The organic layer was dried over sodium sulfate,
filtered and concentrated under reduced pressure. Purification by
column chromatography (ISCO, 12 g silica column, 0-30%
EtOAc/hexane) gave the desired product as a white solid (296 mg,
78%).
[0365] LCMS 438 (M+H).sup.+.
##STR00051##
tert-Butyl
(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)octyl)
carbamate
[0366] Dimethyl
4((8-((tert-butoxycarbonyl)amino)octyl)oxy)phthalate (296 mg, 0.68
mmol, 1 eq.) was dissolved in EtOH (3.4 mL, 0.2 M) followed by
aqueous 3M NaOH (680 .mu.L, 2.04 mmol, 3 eq.). The mixture was
heated to 80.degree. C. for 4 hours. The mixture was allowed to
cool to room temperature, diluted with DCM (12 mL) and 0.5 M HCl
(4.7 mL). The layers were separated and the organic layer was
washed with 6.2 mL water. The aqueous layers were combined and
extracted three times with chloroform (12 mL). The combined organic
layers were dried over sodium sulfate, filtered and condensed to
give the material that was used in next step.
[0367] LCMS 410 (M+H).sup.+.
[0368] The resultant material and 3-aminopiperidine-2,6-dione
hydrochloride (112 mg, 0.68 mmol, 1 eq.) were dissolved in pyridine
(3.4 mL, 0.2 M) and heated to 110.degree. C. overnight. Then the
mixture was cooled to room temperature and concentrated under
reduced pressure, purified by column chromatography (ISCO, 12 g
silica column, 0-7% Methanol/DCM) to give the desired product (170
mg, 50% for two steps).
[0369] LCMS 502 (M+H).sup.+.
##STR00052##
5-((8-Aminooctyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
[0370] tert-Butyl
(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)octyl)
carbamate (170 mg, 0.34 mmol, 1 eq.) was dissolved in TFA (3.4 mL,
0.1 M) and then heated to 50.degree. C. for 3 hours. The mixture
was allowed to cool to room temperature, diluted with MeOH and
concentrated under reduced pressure. The material was purified by
column chromatography (ISCO, 4 g silica column, 0-20% 1.75N
NH.sub.3.MeOH/DCM) to give the free base product (111 mg, 82%).
[0371] LCMS 402 (M+H).sup.+.
##STR00053##
[0372] ZXH-2-45
[0373] To a solution of
(S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,-
2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-carboxylic acid (24 mg,
0.054 mmol, 1 eq.) and
5-((8-aminooctyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
(22 mg, 0.054 mmol, 1 eq.) in DMF (1 mL) were added DIEA (27 .mu.L,
0.16 mmol, 3 eq.) and HATU (25 mg, 0.065 mmol, 1.2 eq.). The
mixture was stirred at room temperature overnight and then purified
by HPLC to give the product as TFA salt (18.3 mg, 36%).
[0374] LCMS 828 (M+H).sup.+.
[0375] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.11 (s, 1H),
8.33 (t, J=5.7 Hz, 1H), 7.82 (d, J=8.3 Hz, 1H), 7.50 (d, J=8.8 Hz,
2H), 7.46 (d, J=8.6 Hz, 2H), 7.41 (d, J=2.2 Hz, 1H), 7.34 (dd,
J=8.4, 2.3 Hz, 1H), 5.12 (dd, J=12.8, 5.4 Hz, 1H), 4.58 (dd, J=7.7,
6.6 Hz, 1H), 4.17 (t, J=6.5 Hz, 2H), 3.68 (s, 3H), 3.47 (qd,
J=16.6, 7.3 Hz, 2H), 3.25 (dq, J=17.2, 6.7 Hz, 2H), 3.17 (s, 1H),
2.90 (ddd, J=16.8, 13.8, 5.4 Hz, 1H), 2.65 (s, 3H), 2.09-2.01 (m,
1H), 1.91 (s, 3H), 1.75 (p, J=6.8 Hz, 2H), 1.53 (t, J=6.9 Hz, 2H),
1.42 (q, J=7.0 Hz, 2H), 1.33 (d, J=3.8 Hz, 6H).
Example 28: Synthesis of ZXH-2-145
##STR00054##
[0376] Dimethyl 3-(2-(tert-butoxy)-2-oxoethoxy)phthalate
[0377] To a solution of 3-Hydroxyphthalic anhydride (1260 mg, 6
mmol, 1 eq.) and tert-butyl 2-bromoacetate (1172 mg, 6 mmol, 1 eq.)
in DMF (10 mL) was added K.sub.2CO.sub.3 (1656 mg, 12 mmol, 2 eq.).
The mixture was stirred at room temperature until the reaction
completed. And then the mixture was diluted with EtOAc and washed
with water and brine. The organic layer was dried over sodium
sulfate, filtered and concentrated under reduced pressure.
Purification by column chromatography (ISCO, 24 g silica column,
0-25% EtOAc/hexane) gave the desired product (1408 mg, 72%).
[0378] LCMS 325 (M+H).sup.+.
##STR00055##
2-(2,3-Bis (methoxycarbonyl)phenoxy)acetic acid
[0379] To a solution of dimethyl
3-(2-(tert-butoxy)-2-oxoethoxy)phthalate (972 mg, 3 mmol) in DCM (6
mL) was added TFA (2 mL). The mixture was then stirred at room
temperature until the reaction completed. And then the mixture was
concentrated under reduced pressure, purification by column
chromatography (ISCO, 24 g silica column, 0-6% MeOH/DCM) gave the
desired product as TFA salt (734 mg, 64%).
[0380] LCMS 269 (M+H).sup.+.
##STR00056##
Dimethyl
3-(2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-2-oxoethoxy)
phthalate
[0381] To a solution of 2-(2,3-bis (methoxycarbonyl)phenoxy)acetic
acid with TFA (382 mg, 1 mmol, 1 eq.) and tert-butyl
(2-aminoethyl)carbamate (160 mg, 1 mmol, 1 eq.) in DMF (5 mL) were
added HATU (456 mg, 1.2 mmol, 1.2 eq.) and DIPEA (495 .mu.L, 3
mmol, 3 eq.), and then the mixture was stirred at room temperature
until the reaction completed. The mixture was then diluted with
EtOAc and washed with water and brine. The organic layer was dried
over sodium sulfate, filtered and concentrated under reduced
pressure. The crude product was used in next step without further
purification.
[0382] LCMS 411 (M+H).sup.+.
##STR00057##
N-(2-Aminoethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl-
)oxy)acetamide
[0383] To a solution of dimethyl
3-(2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-2-oxoethoxy)phthalate
(410 mg, 1 mmol, 1 eq.) in EtOH (5 mL) was added aqueous 3M NaOH (1
mL, 3 mmol, 3 eq.), then the mixture was heated to 80.degree. C.
for 4 hours. The mixture was allowed to cool to room temperature,
diluted with DCM (18 mL) and 0.5M HCl (7.2 mL). The layers were
separated and the organic layer was washed with water (9 mL). The
aqueous layers were combined and extracted three times with
chloroform (18 mL). The combined organic layers were dried over
sodium sulfate, filtered and concentrated to give
N-(2-aminoethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl-
)oxy)acetamide without further purification.
[0384] LCMS 383 (M+H).sup.+.
[0385]
N-(2-aminoethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoli-
n-4-yl)oxy)acetamide and 3-aminopiperidine-2,6-dione hydrochloride
(164 mg, 1 mmol, 1 eq.) were dissolved in pyridine (5 mL, 0.2 M)
and heated to 110.degree. C. overnight. The mixture was cooled to
room temperature, concentrated under reduced pressure, and purified
by column chromatography (ISCO, 12 g silica column, 0-4% MeOH/DCM)
to give the desired product.
[0386] LCMS 473 (M+H).sup.+.
[0387] To a solution of the resultant material (1 mmol, 1 eq.) in
DCM (6 mL) was added TFA (2 mL). The mixture was stirred at room
temperature until the reaction completed. The mixture was
concentrated under reduced pressure and purified by column
chromatography (ISCO, 12 g silica column, 0-30% 1.75N
NH.sub.3.MeOH/DCM) to give the free base product (103 mg, 28% for 4
steps).
[0388] LCMS 373 (M+H).sup.+.
##STR00058##
[0389] ZXH-2-145
[0390] To a solution of
(S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,-
2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-carboxylic acid (20 mg,
0.045 mmol, 1 eq.) and
N-(2-aminoethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl-
)oxy)acetamide (17 mg, 0.045 mmol, 1 eq.) in DMF (1 mL) were added
HATU (21 mg, 0.054 mmol, 1.2 eq.) and DIPEA (22 .mu.L, 0.135 mmol,
3 eq.). The mixture was stirred at room temperature overnight and
then purified by HPLC to give the product as TFA salt (5 mg,
12%).
[0391] LCMS 801 (M+H).sup.+.
[0392] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.11 (s, 1H),
8.32 (d, J=5.5 Hz, 1H), 8.18-8.13 (m, 1H), 7.74 (t, J=7.9 Hz, 1H),
7.52-7.44 (m, 5H), 7.37 (dd, J=8.5, 2.9 Hz, 1H), 5.14-5.08 (m, 1H),
4.62-4.55 (m, 1H), 3.97 (s, 2H), 3.68 (s, 3H), 3.54-3.36 (m, 6H),
2.94-2.86 (m, 1H), 2.64 (s, 3H), 2.60 (d, J=18.0 Hz, 1H), 2.06-1.98
(m, 1H), 1.88 (s, 3H).
Example 29: Synthesis of ZXH-2-147
##STR00059##
[0393]
4-((3-Aminopropyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-
-dione
[0394] To a stirred solution of
2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (277 mg,
1 mmol, 1 eq.) in DMF (5 mL) were added DIPEA (330 .mu.L, 2 mmol, 2
eq.) and tert-butyl (3-aminopropyl)carbamate (191 mg, 1.1 mmol, 1.1
eq.). The reaction mixture was heated to 90.degree. C. overnight.
Cooled to room temperature, the mixture was diluted with EtOAc and
washed with water and brine, dried over Na.sub.2SO.sub.4 and then
concentrated in vacuo give the product that was used in next
step.
[0395] LCMS 431 (M+H).sup.+.
[0396] To a solution of
4-((3-Aminopropyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
(1 mmol, 1 eq.) in DCM (6 mL) was added TFA (2 mL). The mixture was
stirred at room temperature until the reaction completed. And then
the mixture was concentrated under reduced pressure, purified by
column chromatography (ISCO, 12 g silica column, 0-15% 1.75N
NH.sub.3.MeOH/DCM) to give the free base product (236 mg, 72% for 2
steps).
[0397] LCMS 331 (M+H).sup.+.
##STR00060##
[0398] ZXH-2-147
[0399] To a solution of
(S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,-
2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-carboxylic acid (25 mg,
0.056 mmol, 1 eq.) and
4-((3-aminopropyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
(19 mg, 0.056 mmol, 1 eq.) in DMF (1 mL) were added HATU (25 mg,
0.067 mmol, 1.2 eq.) and DIPEA (28 .mu.L, 0.168 mmol, 3 eq.). The
mixture was stirred at room temperature overnight and then purified
by HPLC to give the product as TFA salt (3.5 mg, 8%).
[0400] LCMS 757 (M+H).sup.+.
[0401] .sup.1H NMR (500 MHz, DMSO-.sub.d6) .delta. 11.09 (s, 1H),
8.43-8.36 (m, 1H), 7.61-7.44 (m, 5H), 7.17-7.11 (m, 1H), 7.06 (dd,
J=20.6, 7.8 Hz, 1H), 6.73 (d, J=19.8 Hz, 1H), 5.06 (dd, J=12.7, 5.5
Hz, 1H), 4.58 (ddd, J=8.0, 6.6, 1.3 Hz, 1H), 4.53-4.48 (m, 1H),
3.68 (s, 3H), 3.50-3.44 (m, 3H), 3.34 (d, J=11.6 Hz, 2H), 2.93-2.84
(m, 1H), 2.66 (s, 3H), 2.59 (d, J=19.8 Hz, 1H), 2.42-2.36 (m, 1H),
2.31-2.25 (m, 2H), 2.03 (d, J=7.0 Hz, 1H), 1.93 (s, 3H), 1.82 (p,
J=6.8 Hz, 2H).
Example 30: Synthesis of ZXH-2-184
##STR00061##
[0402]
4-((4-aminobutyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3--
dione
[0403] To a stirred solution of
2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (277 mg,
1 mmol, 1 eq.) in DMF (5 mL) were added DIPEA (330 .mu.L, 2 mmol, 2
eq.) and tert-butyl (4-aminopropyl)carbamate (207 mg, 1.1 mmol, 1.1
eq.). The reaction mixture was heated to 90.degree. C. overnight.
Cooled to room temperature, the mixture was diluted with EtOAc and
washed with water and brine, dried over Na.sub.2SO.sub.4 and
concentrated in vacuo to give the product that was used in next
step.
[0404] LCMS 445 (M+H).sup.+.
[0405] To a solution of
4-((4-aminobutyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
(1 mmol, 1 eq.) in DCM (6 mL) was added TFA (2 mL), then the
mixture was stirred at room temperature until the reaction
completed. The mixture was concentrated under reduced pressure and
purified by column chromatography (ISCO, 12 g silica column, 0-20%
1.75N NH.sub.3.MeOH/DCM) to give the free base product (224 mg, 65%
for 2 steps).
[0406] LCMS 345 (M+H).sup.+.
##STR00062##
[0407] ZXH-2-184
[0408] To a solution of
(S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,-
2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-carboxylic acid (37 mg,
0.08 mmol, 1 eq.) and
4-((4-aminobutyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
(28 mg, 0.08 mmol, 1 eq.) in DMF (1 mL) were added HATU (37 mg, 0.1
mmol, 1.2 eq.) and DIPEA (40 .mu.L, 0.24 mmol, 3 eq.). The mixture
was stirred at room temperature overnight and then purified by HPLC
to give the product as TFA salt (4.8 mg, 7%).
[0409] LCMS 771 (M+H).sup.+.
[0410] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.09 (s, 1H),
8.37 (t, J=5.7 Hz, 1H), 7.56 (dd, J=8.6, 7.1 Hz, 1H), 7.51 (d,
J=8.7 Hz, 2H), 7.46 (d, J=8.6 Hz, 2H), 7.12 (d, J=8.6 Hz, 1H), 7.01
(d, J=7.0 Hz, 1H), 6.59 (t, J=6.1 Hz, 1H), 5.05 (dd, J=12.8, 5.5
Hz, 1H), 4.58 (dd, J=7.7, 6.6 Hz, 1H), 3.68 (s, 3H), 3.47 (qd,
J=16.5, 7.2 Hz, 2H), 3.31 (d, J=5.6 Hz, 2H), 2.89 (ddd, J=17.0,
13.8, 5.4 Hz, 1H), 2.64 (s, 3H), 2.59 (ddd, J=17.0, 4.4, 2.4 Hz,
1H), 2.03 (ddd, J=10.0, 6.2, 2.2 Hz, 1H), 1.90 (s, 3H), 1.61 (q,
J=2.8, 2.4 Hz, 4H), 1.24 (s, 2H).
Example 31: Synthesis of ZXH-3-26
##STR00063##
[0411]
4-((5-Aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-
-dione
[0412] To a stirred solution of
2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (219 mg,
0.8 mmol, 1 eq.) in DMF (4 mL) were added DIPEA (264 .mu.L, 1.6
mmol, 2 eq.) and tert-butyl (5-aminopropyl)carbamate (177 mg, 0.88
mmol, 1.1 eq.). The reaction mixture was heated to 90.degree. C.
overnight. Cooled to room temperature, the mixture was diluted with
EtOAc and EtOAc and washed with water and brine, dried over
Na.sub.2SO.sub.4 and concentrated in vacuo to obtain
4-((5-aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,-
3-dione that was used directly in next step.
[0413] LCMS 458 (M+H).sup.+.
[0414] To a solution of
4-((5-aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
(0.8 mmol, 1 eq.) in DCM (6 mL) was added TFA (2 mL). The mixture
was stirred at room temperature until the reaction completed. The
mixture was concentrated under reduced pressure and purified by
column chromatography (ISCO, 12 g silica column, 0-20% 1.75N
NH.sub.3.MeOH/DCM) to give the free base product (194 mg, 68% for 2
steps).
[0415] LCMS 359 (M+H).sup.+.
##STR00064##
[0416] ZXH-3-26
[0417] To a solution of
(S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,-
2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-carboxylic acid (62 mg,
0.14 mmol, 1 eq.) and
4-((5-aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
(50 mg, 0.14 mmol, 1 eq.) in DMF (1 mL) were added HATU (64 mg,
0.168 mmol, 1.2 eq.) and DIPEA (70 .mu.L, 0.42 mmol, 3 eq.). The
mixture was stirred at room temperature overnight and then purified
by HPLC to give the product as TFA salt (18.4 mg, 15%).
[0418] LCMS 785 (M+H).sup.+.
[0419] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.09 (s, 1H),
8.32 (t, J=5.7 Hz, 1H), 7.57 (dd, J=8.6, 7.1 Hz, 1H), 7.51 (d,
J=8.8 Hz, 2H), 7.46 (d, J=8.6 Hz, 2H), 7.10 (d, J=8.6 Hz, 1H), 7.02
(d, J=7.0 Hz, 1H), 6.55 (t, J=6.0 Hz, 1H), 5.04 (ddd, J=12.8, 5.5,
1.1 Hz, 1H), 4.58 (dd, J=7.7, 6.6 Hz, 1H), 3.68 (s, 3H), 3.47 (qd,
J=16.6, 7.3 Hz, 2H), 3.31-3.23 (m, 4H), 2.89 (ddd, J=16.9, 13.8,
5.5 Hz, 1H), 2.65 (s, 3H), 2.59 (ddd, J=17.0, 4.4, 2.5 Hz, 1H),
2.05-1.99 (m, 1H), 1.90 (s, 3H), 1.60 (dp, J=21.6, 7.2 Hz, 4H),
1.40 (h, J=7.4, 6.5 Hz, 2H).
Example 32: Synthesis of ZXH-3-27
##STR00065##
[0420]
4-((2-Aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3--
dione
[0421] To a stirred solution of
2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (180 mg,
0.65 mmol, 1 eq.) in DMF (4 mL) were added DIPEA (214 .mu.L, 1.3
mmol, 2 eq.) and tert-butyl (2-aminopropyl)carbamate (114 mg, 0.72
mmol, 1.1 eq.). The reaction mixture was heated to 90.degree. C.
overnight. Cooled to room temperature, the mixture was diluted with
EtOAc and washed with water and brine, dried over Na.sub.2SO.sub.4
and concentrated in vacuo and then used in next step without
further purification.
[0422] LCMS 417 (M+H).sup.+.
[0423] To a solution of
4-((2-aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
(0.65 mmol, 1 eq.) dissolved in DCM (6 mL) was added TFA (2 mL),
then the mixture was stirred at room temperature until the reaction
completed. The mixture was concentrated under reduced pressure and
purified by column chromatography (ISCO, 12 g silica column, 0-15%
1.75N NH.sub.3.MeOH/DCM) to give the free base product (100 mg, 49%
for 2 steps).
[0424] LCMS 317 (M+H).sup.+.
##STR00066##
[0425] ZXH-3-27
[0426] To a solution of
(S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,-
2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-carboxylic acid (44 mg,
0.1 mmol, 1 eq.) and
4-((2-aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
(31 mg, 0.1 mmol, 1 eq.) in DMF (1 mL) were added HATU (46 mg, 0.12
mmol, 1.2 eq.) and DIPEA (50 .mu.L, 0.3 mmol, 3 eq.). The mixture
was stirred at room temperature overnight and then purified by HPLC
to give the product as TFA salt (6.6 mg, 8%).
[0427] LCMS 743 (M+H).sup.+.
[0428] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.09 (s, 1H),
8.49 (dt, J=5.7, 2.9 Hz, 1H), 7.61 (ddd, J=8.6, 7.0, 1.6 Hz, 1H),
7.54-7.48 (m, 2H), 7.45 (d, J=8.4 Hz, 2H), 7.24 (d, J=8.6 Hz, 1H),
7.04 (d, J=7.1 Hz, 1H), 6.83 (t, J=6.0 Hz, 1H), 5.05 (ddd, J=12.8,
5.5, 1.4 Hz, 1H), 4.58 (ddd, J=7.6, 6.6, 0.9 Hz, 1H), 3.68 (s, 3H),
3.57-3.42 (m, 6H), 2.89 (ddd, J=17.8, 13.9, 5.4 Hz, 1H), 2.64 (d,
J=1.1 Hz, 3H), 2.59 (ddd, J=15.0, 4.7, 2.4 Hz, 1H), 2.01 (dtd,
J=12.5, 5.2, 2.2 Hz, 1H), 1.90 (d, J=2.6 Hz, 3H).
Example 33: Synthesis of ZXH-3-028
##STR00067##
[0429]
5-((2-aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3--
dione
[0430] To a solution of
2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (50 mg,
0.18 mmol) in DMF (1 mL) were added tert-butyl
(2-aminoethyl)carbamate (29 mg, 0.18 mmol) and DIEA (89 .mu.L, 0.54
mmol). The mixture was stirred at 120.degree. C. for 1 h, and the
crude product was purified by HPLC (MeOH/H.sub.2O, 0.035% TFA) to
give intermediate
5-((2-aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione.
The intermediate was dissolved in TFA/DCM (4 mL, v/v=1/3), stirred
for 1 h, and then concentrated in vacuo to give the product as TFA
salt (46 mg, 80% for 2 steps).
[0431] LCMS: 317 (M+H).sup.+.
##STR00068##
[0432] ZXH-3-028
[0433] To a solution of
(S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,-
2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-carboxylic acid (73 mg,
0.16 mmol) and
5-((2-aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline--
1,3-dione (TFA salt, 46 mg, 0.11 mmol) in DMF (1 mL) were added
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (32 mg, 0.16
mmol), 1-hydroxybenzotriazole (HOBt) (22 mg, 0.16 mmol) and DMAP
(20 mg, 0.16 mmol). The mixture was stirred at room temperature
overnight and then purified by HPLC (MeOH/H.sub.2O, 0.035% TFA) to
give ZXH-3-028 as TFA salt (3 mg, 3%).
[0434] LCMS: 743 (M+H).sup.+.
[0435] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.05 (d, J=4.5
Hz, 1H), 8.40 (s, 1H), 7.59 (d, J=8.3 Hz, 1H), 7.51 (d, J=8.9 Hz,
1H), 7.45 (d, J=8.5 Hz, 2H), 7.27 (s, 1H), 7.05 (d, J=2.1 Hz, 1H),
6.92 (dd, J=8.4, 2.1 Hz, 1H), 5.03 (ddd, J=12.8, 5.5, 2.4 Hz, 1H),
4.58 (t, J=7.2 Hz, 1H), 3.68 (s, 3H), 3.51-3.44 (m, 2H), 2.92-2.83
(m, 1H), 2.65 (d, J=1.9 Hz, 3H), 2.61-2.55 (m, 1H), 2.55 (s, 1H),
2.19 (t, J=7.4 Hz, 1H), 2.02-1.96 (m, 1H), 1.93 (d, J=2.8 Hz, 3H),
1.48 (t, J=7.4 Hz, 1H).
Example 34: Synthesis of ZXH-3-195
##STR00069##
[0436]
5-((5-Aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-
-dione
[0437] To a solution of
2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (83 mg,
0.3 mmol) in DMF (1 mL) were added tert-butyl
(5-aminopentyl)carbamate (61 mg, 0.3 mmol) and DIEA (148 .mu.L, 0.9
mmol). The mixture was stirred at 120.degree. C. for 1 h, and the
crude was purified by HPLC (MeOH/H.sub.2O, 0.035% TFA) to give the
intermediate. The intermediate was dissolved in TFA/DCM (4 mL,
v/v=1/3) and then concentrated in vacuo to give the product as TFA
salt (79 mg, 56% for 2 steps).
[0438] LCMS: 359 (M+H).sup.+.
##STR00070##
[0439] ZXH-3-195
[0440] To a solution of
(S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,-
2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-carboxylic acid (13 mg,
0.03 mmol) and
5-((5-aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-
-1,3-dione (14 mg, 0.03 mmol) in DMF (1 mL) were added HATU (14 mg,
0.036 mmol) and DIPEA (25 .mu.L, 0.15 mmol). The mixture was
stirred at room temperature for 1 h and then purified by HPLC
(MeOH/H.sub.2O, 0.035% TFA) to give ZXH-3-195 as TFA salt (7.9 mg,
29%).
[0441] LCMS: 785 (M+H).sup.+.
[0442] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.06 (s, 1H),
8.31 (dt, J=6.1, 3.0 Hz, 1H), 7.55 (d, J=8.4 Hz, 1H), 7.51 (d,
J=8.8 Hz, 2H), 7.46 (d, J=8.6 Hz, 2H), 7.11 (t, J=5.4 Hz, 1H), 6.95
(d, J=2.1 Hz, 1H), 6.84 (dd, J=8.4, 2.1 Hz, 1H), 5.03 (dd, J=12.8,
5.4 Hz, 1H), 4.58 (dd, J=7.8, 6.6 Hz, 1H), 3.68 (s, 3H), 3.53-3.43
(m, 2H), 3.30-3.26 (m, 2H), 3.17 (q, J=6.4, 5.2 Hz, 2H), 2.88 (ddd,
J=16.8, 13.7, 5.4 Hz, 1H), 2.65 (s, 3H), 2.61-2.54 (m, 1H),
2.03-1.95 (m, 1H), 1.91 (s, 3H), 1.60 (dp, J=21.4, 7.1 Hz, 4H),
1.43 (dt, J=11.8, 7.3 Hz, 2H).
Example 35: Synthesis of ZXH-3-142
##STR00071##
[0443] N-(2,6-Dioxopiperidin-3-yl)-6-nitrohexanamide
[0444] A solution of 6-nitrohexanoic acid (260 mg, 1.61 mmol) in
thionyl chloride (3 mL) was stirred at 90.degree. C. for 1 h and
then was concentrated in vacuo to obtain the intermediate
6-nitrohexanoyl chloride. The intermediate was then dissolved in
DCM (2 mL), and then added into the solution of
3-aminopiperidine-2,6-dione hydrochloride salt (266 mg, 1.61 mmol)
and DIEA (1.3 mL, 8.05 mmol) in DCM (2 mL) at 0.degree. C. The
mixture was then stirred at room temperature for 3 h, and then
purified by HPLC (MeOH/H.sub.2O, 0.035% TFA) to give the product as
TFA salt.
[0445] LCMS: 272 (M+H).sup.+.
##STR00072##
6-Amino-N-(2,6-dioxopiperidin-3-yl)hexanamide
[0446] N-(2,6-Dioxopiperidin-3-yl)-6-nitrohexanamide from last step
was dissolved in MeOH and then hydrogenation was conducted to give
the product (111 mg, 29% for 2 steps).
[0447] LCMS: 242 (M+H).sup.+.
##STR00073##
[0448] ZXH-3-142
[0449] To a solution of perfluorophenyl
(S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,-
2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-carboxylate (73 mg, 0.12
mmol) and 6-amino-N-(2,6-dioxopiperidin-3-yl)hexanamide (30 mg,
0.12 mmol) in DMF (1 mL) was added 4-pyrrolidinopyridine (71 mg,
0.48 mmol), the mixture was stirred at room temperature for 3 h,
and then purified by HPLC (MeOH/H.sub.2O, 0.035% TFA) to give the
product as TFA salt (1.7 mg, 2%).
[0450] LCMS: 668 (M+H).sup.+.
Example 36: Synthesis of ZXH-3-052
##STR00074##
[0451] tert-Butyl
(3-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)prop-2-yn-1-yl)c-
arbamate
[0452] To a solution of
4-bromo-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (758 mg,
2.26 mmol) and tert-butyl prop-2-yn-1-ylcarbamate (700 mg, 4.5
mmol) in DMF (10 mL) were added CuI (86 mg, 0.45 mmol),
Pd(pph3)2Cl.sub.2 (158 mg, 0.226 mmol) and Et3N (5.6 mL). The
mixture was then stirred at 70.degree. C. for 3 h. The reaction was
allowed to cool to room temperature and then filtered. The filtrate
was concentrated in vacuo and then purified by flash column
chromatography with silica gel (MeOH/DCM, 0-4%) to obtain the
desired product with trace DMF.
[0453] LCMS: 412 (M+H).sup.+.
##STR00075##
4-(3-Aminopropyl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
[0454] tert-Butyl
(3-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)prop-2-yn-1-yl)c-
arbamate from last steps was dissolved in MeOH, and then
hydrogenation was conducted to obtain the intermediate. The
intermediate was then dissolved in TFA/DCM (4 mL, v/v=1/3), stirred
at room temperature for 3 h, and then purified by HPLC
(MeOH/H.sub.2O, 0.035% TFA) to give product as TFA salt (300 mg,
31% for 3 steps).
[0455] LCMS: 315 (M+H).sup.+.
##STR00076##
[0456] ZXH-3-052
[0457] To a solution of
(S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,-
2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-carboxylic acid (22 mg,
0.05 mmol) and
4-(3-aminopropyl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-di-
one (20 mg, 0.045 mmol) in DMF (1 mL) were added HATU (21 mg, 0.054
mmol) and DIEA (22 uL, 0.135 mmol). The mixture was stirred at room
temperature for 1 h, and the crude product was purified by HPLC
(MeOH/H.sub.2O, 0.035% TFA) to give ZXH-3-052 as TFA salt (19.5 mg,
51%).
[0458] LCMS: 742 (M+H).sup.+.
[0459] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.12 (s, 1H),
8.38 (dt, J=6.3, 3.1 Hz, 1H), 7.87-7.80 (m, 2H), 7.75 (dd, J=7.7,
1.5 Hz, 1H), 7.51 (d, J=8.8 Hz, 2H), 7.46 (d, J=8.6 Hz, 2H), 5.14
(dd, J=12.8, 5.5 Hz, 1H), 4.58 (dd, J=7.8, 6.7 Hz, 1H), 3.68 (s,
3H), 3.47 (qd, J=16.6, 7.1 Hz, 2H), 3.33-3.25 (m, 2H), 2.95-2.88
(m, 1H), 2.88-2.80 (m, 2H), 2.66 (s, 3H), 2.64-2.58 (m, 1H), 2.55
(s, 1H), 2.05 (dtd, J=12.9, 5.3, 2.2 Hz, 1H), 1.93 (d, J=1.1 Hz,
3H), 1.92-1.88 (m, 2H).
Example 37: Synthesis of Methyl
2-((6S)-4-(4-chlorophenyl)-2-((5-((2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1-
-oxoisoindolin-4-yl)amino)pentyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][-
1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (BJG-01-174)
##STR00077##
[0460] Methyl 2-(bromomethyl)-5-fluoro-3-nitrobenzoate
[0461] Methyl 2-methyl-5-fluoro-3-nitrobenzoate (500 mg, 2.35 mmol,
1 eq.), N-bromosuccinimide (NBS) (520 mg, 2.85 mmol, 1.2 eq.), and
Azobisisobutyronitrile (AIBN) (54.6 mg, 0.33 mmol, 0.15 eq.) were
dissolved in benzene (6 mL, 0.4 M). The reaction was sparged with
nitrogen for 15 minutes, and then heated to 80.degree. C. for 21
hours. The reaction was cooled to room temperature and diluted with
EtOAc (50 mL). The organic layer was washed sequentially with
water, saturated NaHCO.sub.3, and brine. The organic layer was
dried over magnesium sulfate, filtered, and concentrated under
reduced pressure to provide a clear, yellow oil. NMR analysis
showed a 4:1 mixture of methyl
2-(bromomethyl)-5-fluoro-3-nitrobenzoate and remaining starting
material (RSM) 2-methyl-5-fluoro-3-nitrobenzoate. The inseparable
mixture was subjected directly to the lactamization reaction.
[0462] .sup.1H NMR: (500 MHz, CDCl.sub.3) .delta. 7.85 (dd, J=8.1,
2.8 Hz, 1H), 7.71 (dd, J=7.3, 2.8 Hz, 1H), 5.12 (s, 2H), 4.01 (s,
3H).
[0463] LC-MS: 314.07/316.07 (M+H).sup.+.
[0464] TLC: R.sub.f=0.6, 2:1 hexanes/EtOAc.
##STR00078##
3-(6-Fluoro-4-nitro-1-oxoisoindolin-2-yl)piperidine-2,6-dione
[0465] To the crude mixture of methyl
2-(bromomethyl)-5-fluoro-3-nitrobenzoate and methyl
2-methyl-5-fluoro-3-nitrobenzoate (2.35 mmol, 1 eq.) were added
K.sub.2CO.sub.3 (816 mg, 5.88 mmol, 2.5 eq.),
3-aminopiperidine-2,6-dione hydrochloride (580 mg, 3.55 mmol, 1.5
eq.), and DMF (4.0 mL, 0.7 M). The reaction was heated to
60.degree. C. for 14 hours, and then cooled to room temperature.
Water (4 mL) was added to precipitate the product, and the
suspension was stirred for 1 hour. The product was collected by
suction filtration, washed with water (25 mL) and DCM (10 mL), and
dried under vacuum to provide a blue-gray solid (331 mg, 45% yield
over 2 steps). The crude product was pure by .sup.1H-NMR and LC-MS
analyses and did not require additional purification.
[0466] .sup.1H NMR: (500 MHz, DMSO-d.sub.6) .delta. 11.03 (s, 1H),
8.33 (dd, J=8.8, 2.4 Hz, 1H), 8.06 (dd, J=6.9, 2.3 Hz, 1H), 5.18
(dd, J=13.3, 5.2 Hz, 1H), 4.87 (d, J=19.0 Hz, 1H), 4.78 (d, J=19.0
Hz, 1H), 2.96-2.85 (m, 1H), 2.65-2.58 (m, 1H), 2.53 (m, 1H), 2.03
(ddd, J=11.6, 6.2, 4.2 Hz, 1H).
##STR00079##
3-(4-Amino-6-fluoro-1-oxoisoindolin-2-yl)piperidine-2,6-dione
[0467] A suspension of
3-(6-Fluoro-4-nitro-1-oxoisoindolin-2-yl)piperidine-2,6-dione (277
mg, 0.90 mmol, 1 eq.) and palladium on charcoal (10 wt %; 98.2 mg,
0.09 mmol, 0.10 eq.) in THF (5 mL, 0.18 M) was sparged with H.sub.2
for 10 minutes. The reaction was stirred overnight under a H.sub.2
balloon. After 14 hours, the reaction was diluted with methanol (25
mL), filtered through Celite, and concentrated under reduced
pressure to provide the desired product as a gray solid (131.2 mg,
52% yield). The material was 95% pure by .sup.1H-NMR and did not
require further purification.
[0468] .sup.1H NMR: (500 MHz, DMSO-d.sub.6) .delta. 10.99 (s, 1H),
6.59 (dd, J=7.7, 2.2 Hz, 1H), 6.54 (dd, J=11.8, 2.3 Hz, 1H), 5.71
(s, 2H), 5.09 (dd, J=13.3, 5.1 Hz, 1H), 4.17 (d, J=16.9 Hz, 1H),
4.09 (d, J=16.8 Hz, 1H), 2.90 (ddd, J=17.4, 13.9, 5.4 Hz, 1H), 2.61
(d, J=17.3 Hz, 1H), 2.29 (qd, J=13.3, 4.5 Hz, 1H), 2.04 (m,
1H).
[0469] LC-MS: 278.17 (M+H).
##STR00080##
tert-Butyl
(5-((2-((2,6-dioxopiperidin-3-yl)-6-fluoro-1-oxoisoindolin-4-yl)amino)pen-
tyl)carbamate
[0470]
3-(4-Amino-6-fluoro-1-oxoisoindolin-2-yl)piperidine-2,6-dione (84.5
mg, 0.30 mmol, 1.2 eq.), tert-butyl (5-bromopentyl)carbamate (66.2
mg, 0.25 mmol, 1.0 eq.), and K.sub.2CO.sub.3 (67.0 mg, 0.50 mmol,
2.0 eq.) were dissolved in DMF (1.25 mL, 0.2 M) and the mixture was
heated to 50.degree. C. After 14 hours, the reaction was cooled and
quenched with EtOAc and water (3 mL each). The aqueous layer was
extracted three times with EtOAc (3 mL). The combined organic
layers were washed with water and then brine, dried over magnesium
sulfate, filtered, and concentrated under reduced pressure.
Purification by column chromatography (ISCO, 12 g silica column,
0-15% MeOH/DCM, 10 minute gradient) provided the desired product as
a clear yellow oil (40.5 mg, 35% yield).
[0471] .sup.1H NMR: (500 MHz, MeOH-d.sub.4) .delta. 6.74 (dd,
J=7.7, 2.6 Hz, 1H), 6.61 (dd, J=11.4, 2.4 Hz, 1H), 5.12 (dd,
J=13.2, 5.5 Hz, 1H), 4.28 (d, J=16.8 Hz, 1H), 4.22 (d, J=16.6 Hz,
1H), 3.82-3.70 (m, 2H), 3.02 (q, J=6.6 Hz, 2H), 2.96-2.88 (m, 1H),
2.42 (qd, J=13.0, 6.0 Hz, 1H), 2.16 (m, 1H), 1.53 (quint, J=7.8 Hz,
2H), 1.48 (m, 1H), 1.42 (s, 9H), 1.34-1.27 (m, 2H).
[0472] LC-MS: 485.28 (M+Na).sup.+, 463.28 (M+H).sup.+, 363.17
(M-Boc).sup.+.
[0473] TLC: R.sub.f=0.4, 10% MeOH/DCM.
##STR00081##
3-(4-((5-Aminopentyl)amino)-6-fluoro-1-oxoisoindolin-2-yl)piperidine-2,6--
dione trifluoroacetate salt
[0474] tert-Butyl
(5-((2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1-oxoisoindolin-4-yl)amino)pent-
yl)carbamate (40.5 mg, 0.088 mmol, 1 eq.) was dissolved in 1:1
DCM/TFA (1.0 mL) and stirred at 50.degree. C. After 2 hours,
solvents were removed under reduced pressure. The product was
lyophilized to give a tan solid (46.9 mg, quantitative yield).
LC-MS analysis showed full Boc-deprotection.
[0475] LC-MS: 363.27 (M+H).sup.+.
##STR00082##
[0476] BJG-01-174
[0477]
3-(4-((5-aminopentyl)amino)-6-fluoro-1-oxoisoindolin-2-yl)piperidin-
e-2,6-dione trifluoroacetate salt (16.7 mg, 0.036 mmol, 1.2 eq.),
(S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,-
2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-carboxylic acid (13.1
mg, 0.030 mmol, 1.0 eq.), and HATU (13.4 mg, 0.036 mmol, 1.2 eq.)
were dissolved in DMSO (0.50 mL, 0.06 M). DIPEA (20.70 .mu.L, 0.119
mmol, 4 eq.) was added. The reaction was stirred at room
temperature for 4 hours, and then diluted with MeOH (1 mL) and
purified by reverse-phase prep HPLC (100-0% H.sub.2O/MeOH, 45
minute gradient). The product was lyophilized from H.sub.2O/MeCN to
provide a white powder (7.9 mg, TFA salt, 29% yield).
[0478] .sup.1H NMR: (500 MHz, DMSO-d.sub.6) .delta. 8.31 (t, J=5.7
Hz, 1H), 7.49 (d, J=8.4 Hz, 2H), 7.45 (d, J=8.4 Hz, 2H), 6.61 (d,
J=7.7, 1H), 6.56 (d, J=11.7 Hz, 1H), 5.79 (s, 1H), 5.15 (dd,
J=13.6, 5.1 Hz, 1H), 4.56 (td, J=7.2, 2.1 Hz, 1H), 4.18 (dd,
J=16.9, 5.9 Hz, 1H), 4.06 (dd, J=16.9, 2.2 Hz, 1H), 3.67 (s, 3H),
3.62 (m, 2H), 3.52-3.42 (m, 3H), 3.26-3.19 (m, 2H), 2.99 (ddd,
J=18.1, 13.5, 5.4 Hz, 1H), 2.75 (d, J=17.7, Hz, 1H), 2.63 (s, 3H),
2.26 (m, 1H), 2.09-2.00 (m, 1H), 1.90 (s, 3H), 1.51 (quint, J=7.5
Hz, 2H), 1.46 (quint, J=7.6 Hz, 2H), 1.27 (m, 2H).
[0479] LC-MS: 789.31 (M+H).sup.+.
Example 38: Synthesis of Methyl
2-((6S)-4-(4-chlorophenyl)-2-((5-((2-(2,6-dioxopiperidin-3-yl)-7-fluoro-1-
,3-dioxoisoindolin-4-yl)amino)pentyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-
f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (BJG-02-119)
##STR00083##
[0480]
2-(2,6-Dioxopiperidin-3-yl)-4,7-difluoroisoindoline-1,3-dione
[0481] 3,6-Difluorophthalic anhydride (77.3 mg, 0.40 mmol, 1.0
eq.), potassium acetate (120.8 mg, 1.24 mmol, 3.1 eq.), and
3-aminopiperidine-2,6-dione hydrochloride (80.4 mg, 0.48 mmol, 1.2
eq.) were dissolved in glacial acetic acid (1.2 mL, 0.33 M), and
then the mixture was heated to 120.degree. C. After 16 hours, the
reaction was cooled to room temperature and the excess acetic acid
was removed by rotary evaporation. The residue was dissolved in
EtOAc and water (20 mL each), and the aqueous layer was extracted 4
times with EtOAc (15 mL). The combined organic layers were washed
with water and then brine, dried over magnesium sulfate, filtered,
and concentrated under reduced pressure to provide the desired
product as a tan solid (94.0 mg, 80% yield).
[0482] .sup.1H NMR: (500 MHz, DMSO-d.sub.6) .delta. 11.14 (s, 1H),
7.79 (t, J=5.7 Hz, 2H), 5.15 (dd, J=12.9, 5.4 Hz, 1H), 2.88 (ddd,
J=17.1, 13.8, 5.5 Hz, 1H), 2.60 (d, J=17.3 Hz, 1H), 2.55-2.45 (m,
1H), 2.05 (m, 1H).
[0483] LC-MS: 295.17 (M+H).sup.+.
##STR00084##
tert-Butyl
(5-((2-(2,6-dioxopiperidin-3-yl)-7-fluoro-1,3-dioxoisoindolin-4-yl)amino)-
pentyl)carbamate
[0484]
2-(2,6-Dioxopiperidin-3-yl)-4,7-difluoroisoindoline-1,3-dione (58.8
mg, 0.20 mmol, 1.0 eq.) and tert-butyl (5-aminopentyl)carbamate
(44.4 mg, 0.22 mmol, 1.1 eq.) were dissolved in DMSO (0.7 mL, 0.3
M). DIPEA (0.070 mL, 0.40 mmol, 2.0 eq.) was added at room
temperature, and the solution was heated to 130.degree. C. for 2
hours. The reaction was then cooled to room temperature and
quenched with water (3 mL). The aqueous layer was extracted 4 times
with EtOAc (15 mL). The combined organic layers were washed three
times with water, then once with brine, dried over magnesium
sulfate, filtered, and concentrated under reduced pressure. The
crude material, a dark green oil, was carried on directly to the
Boc-deprotection reaction.
[0485] LC-MS: 499.28 (M+Na).sup.+, 377.27 (M-Boc).sup.+.
##STR00085##
4-((5-aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)-7-fluoroisoindoline--
1,3-dione trifluoroacetate salt
[0486] Crude tert-butyl
(5-((2-(2,6-dioxopiperidin-3-yl)-7-fluoro-1,3-dioxoisoindolin-4-yl)amino)-
pentyl)carbamate (0.20 mmol) was dissolved in 4:1 DCM/TFA (2.5 mL,
0.08 M) and heated to 50.degree. C. After 2 hours, the reaction was
cooled to room temperature and concentrated under reduced pressure.
Reverse-phase prep HPLC (100-40% H.sub.2O/MeCN, 60-minute
gradient), followed by lyophilization from MeCN/water provided the
desired product as a forest-green powder (53.7 mg, 54% yield over 2
steps).
[0487] LC-MS: 377.07 (M+H).sup.+.
##STR00086##
[0488] (BJG-02-119)
[0489]
4-((5-aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)-7-fluoroisoind-
oline-1,3-dione trifluoroacetate salt (18.1 mg, 0.033 mmol, 1.1
eq.),
(S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,-
2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-carboxylic acid (13.4
mg, 0.030 mmol, 1.0 eq.), and HATU (12.9 mg, 0.033 mmol, 1.1 eq.)
were dissolved in DMSO (0.60 mL, 0.05 M). DIPEA (16.00 .mu.L, 0.092
mmol, 3 eq.) was added. The reaction was stirred at room
temperature overnight, and then diluted with DMSO (1 mL). The crude
product purified by reverse-phase prep HPLC (100-0% H.sub.2O/MeCN,
45 minute gradient). The product was lyophilized from H.sub.2O/MeCN
to provide a yellow powder (15.6 mg, TFA salt, 56% yield).
[0490] .sup.1H NMR: (500 MHz, DMSO-d.sub.6) .delta. 11.10 (s, 1H),
8.31 (t, J=5.7 Hz, 1H), 7.50 (d, J=8.8 Hz, 2H), 7.47-7.42 (m, 3H),
7.16 (dd, J=9.4, 3.3 Hz, 1H), 6.50 (s, 1H), 5.04 (dd, J=12.8, 5.4
Hz, 1H), 4.57 (t, J=7.2 Hz, 1H), 3.67 (s, 3H), 3.50 (dd, J=16.6,
6.7 Hz, 1H), 3.43 (dd, J=16.6, 7.8 Hz, 1H), 3.33-3.19 (m, 4H), 2.87
(ddd, J=17.1, 13.9, 5.4 Hz, 1H), 2.64 (s, 3H), 2.62-2.55 (m, 1H),
2.05-1.97 (m, 1H), 1.90 (d, J=1.2 Hz, 3H), 1.57 (m, 4H), 1.42-1.33
(m, 2H).
[0491] .sup.19F NMR: (471 MHz, DMSO-d.sub.6) .delta. -130.4 (aryl
fluoride), -74.7 (TFA).
[0492] LC-MS: 803.51 (M+H).sup.+.
Example 39: Synthesis of Methyl
2-((6S)-4-(4-chlorophenyl)-2-((5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoi-
soindolin-4-yl)oxy)pentyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]t-
riazolo[4,3-a][1,4]diazepin-6-yl)acetate (BJG-02-030)
##STR00087##
[0493]
2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione
[0494] 3-Hydroxyphthalic anhydride (329 mg, 2.0 mmol, 1.0 eq.),
potassium acetate (617 mg, 6.2 mmol, 3.1 eq.), and
3-aminopiperidine-2,6-dione hydrochloride (375 mg, 2.3 mmol, 1.15
eq.) were dissolved in glacial acetic acid (6.0 mL, 0.33 M), and
then the mixture was heated to 120.degree. C. After 16 hours, the
reaction was cooled to room temperature and acetic acid was removed
by rotary evaporation. The residue was dissolved in EtOAc and water
(20 mL each), and the aqueous layer was extracted 5 times with
EtOAc (40 mL). The combined organic layers were washed twice with
water and then brine, dried over magnesium sulfate, filtered, and
concentrated to provide the desired product as a red powder (390
mg, 71% yield). The material was >95% pure by 1H-NMR and did not
require further purification.
[0495] .sup.1H NMR: (500 MHz, DMSO-d.sub.6) .delta. 11.16 (s, 1H),
11.08 (s, 1H), 7.65 (t, J=7.8 Hz, 1H), 7.32 (d, J=7.2 Hz, 1H), 7.25
(d, J=8.4 Hz, 1H), 5.07 (dd, J=12.8, 5.4 Hz, 1H), 2.88 (ddd,
J=16.9, 13.8, 5.5 Hz, 1H), 2.58 (d, J=18.4 Hz, 1H), 2.53 (m, 1H),
2.06-1.98 (m, 1H).
[0496] LC-MS: 275.07 (M+H).sup.+.
##STR00088##
tert-Butyl
(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)pentyl)
carbamate
[0497] 2-(2,6-Dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione
(54.7 mg, 0.20 mmol, 1.0 eq.), tert-butyl (5-bromopentyl)carbamate
(64.0 mg, 0.24 mmol, 1.2 eq.), and K.sub.2CO.sub.3 (54.6 mg, 0.40
mmol, 2.0 eq.) were dissolved in DMF (0.5 mL, 0.4 M) and heated to
50.degree. C. After 18 hours, the reaction was cooled and quenched
with EtOAc and water (3 mL each). The aqueous layer was extracted 4
times with EtOAc (10 mL). The combined organic layers were washed
with water and then brine, dried over magnesium sulfate, filtered,
and concentrated under reduced pressure. Purification by column
chromatography (ISCO, 12 g silica column, 0-15% MeOH/DCM, 10 minute
gradient) provided the desired product as a viscous yellow oil in
quantitative yield.
[0498] LC-MS: 482.28 (M+Na).sup.+, 360.27 (M-Boc).sup.+.
[0499] TLC: R.sub.f=0.33, 5% MeOH/DCM
##STR00089##
4-((5-Aminopentyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
trifluoroacetate salt
[0500] tert-Butyl
(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)pentyl)carb-
amate (0.20 mmol) was dissolved in 1.5:1 DCM/TFA (1.25 mL, 0.16 M)
and heated to 50.degree. C. After 2 hours, the reaction was cooled
to room temperature and concentrated under reduced pressure.
Lyophilization from MeCN/water provided the desired product as a
sticky, colorless oil (82.4 mg, 87% yield).
[0501] .sup.1H NMR: (500 MHz, DMSO-d.sub.6) .delta. 11.13 (s, 1H),
7.88-7.82 (m, 1H), 7.54 (t, J=8.9 Hz, 1H), 7.48 (d, J=7.3 Hz, 1H),
5.10 (dd, J=12.8, 5.4 Hz, 1H), 4.24 (t, J=6.1 Hz, 2H), 3.05-2.94
(m, 1H), 2.90-2.77 (m, 2H), 2.69-2.60 (m, 1H), 2.12-2.00 (m, 2H),
1.83 (p, J=6.6 Hz, 2H), 1.65 (p, J=7.4 Hz, 2H), 1.60-1.51 (m,
2H).
[0502] LC-MS: 360.27 (M+H).sup.+.
##STR00090##
[0503] BJG-02-030
[0504]
4-((5-aminopentyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-d-
ione trifluoroacetate salt (17.2 mg, 0.036 mmol, 1.2 eq.),
(S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,-
2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-carboxylic acid (14.1
mg, 0.030 mmol, 1.0 eq.), and HATU (15.0 mg, 0.039 mmol, 1.3 eq.)
were dissolved in DMSO (0.50 mL, 0.06 M). DIPEA (18.00 .mu.L, 0.103
mmol, 3.4 eq.) was added. The reaction was stirred at room
temperature overnight, and then diluted with DMSO (1 mL) and
purified by reverse-phase prep HPLC (100-0% H.sub.2O/MeCN, 45
minute gradient). The product was lyophilized from H.sub.2O/MeCN to
provide a white powder (7.9 mg, TFA salt, 29% yield).
[0505] .sup.1H NMR: (500 MHz, DMSO-d.sub.6) .delta. 11.09 (s, 1H),
8.33 (t, J=5.7 Hz, 1H), 7.80 (t, J=7.9 Hz, 1H), 7.53-7.48 (m, 3H),
7.46-7.41 (m, 3H), 5.06 (dd, J=12.8, 5.4 Hz, 1H), 4.57 (t, J=7.2
Hz, 1H), 4.21 (t, J=6.4 Hz, 2H), 3.46 (qd, J=16.6, 7.2 Hz, 2H),
3.28 (dp, J=19.7, 6.5 Hz, 2H), 2.87 (ddd, J=17.1, 13.8, 5.4 Hz,
1H), 2.64 (s, 3H), 2.58 (d, J=17.6 Hz, 1H), 2.07-1.97 (m, 1H), 1.90
(s, 3H), 1.80 (p, J=6.7 Hz, 2H), 1.61 (p, J=7.1 Hz, 2H), 1.51 (p,
J=8.0 Hz, 2H).
[0506] LC-MS: 786.41 (M+H).sup.+.
[0507] Tables
TABLE-US-00001 TABLE 1 Data collection and refinement statistics.
DDB1.DELTA.B-CRBN- DDB1.DELTA.B-CRBN- DDB1.DELTA.B-CRBN- dBET55-
dBET6-BRD4BD1 dBET23-BRD4BD1 BRD4BD1 D145A Data collection Space
group P 65 2 2 P 65 2 2 P 65 2 2 Cell dimensions a, b, c (.ANG.)
115.40, 115.40, 588.14 115.57, 115.57, 596.32 115.204, 115.20,
597.14 .alpha., .beta., .gamma. (.degree.) 90, 90, 120 90, 90, 120
90, 90, 120 Resolution (.ANG.) 49.7-3.3 49.9-3.5 99.8-3.9 (3.4-3.3)
(3.6-3.5) (4.1-4.0) R.sub.merge 0.0201 (0.6794) 0.0263 (0.7619)
0.3213 (2.743) I/.sigma. I 17.83 (0.96) 12.73 (0.88) 8.20 (0.86)
Completeness (%) 99.48 (95.66) 97.82 (88.45) 99.95 (99.71)
Redundancy 2.0 (2.0) 2.0 (2.0) 17.4 (16.2) Refinement Resolution
(.ANG.) 3.3 3.5 3.9 No. reflections 35240 (3287) 30444 (2658) 21193
(2038) R.sub.work 0.2167 (0.3428) 0.2292 (0.3561) 0.2888 (0.3749)
R.sub.free 0.2441 (0.3725) 0.2506 (0.4034) 0.3087 (0.3917) No.
atoms Protein 1289 10267 10256 Ligand/ion 60 63 1 Water 0 0 0
B-factors Protein 182.91 208.13 226.95 Ligand/ion 143.02 208.40
131.82 Water -- -- -- R.m.s. deviations Bond lengths (.ANG.) 0.015
0.014 0.012 Bond angles (.degree.) 1.81 1.84 1.75 Each dataset was
collected from one crystal. Values in parentheses are for
highest-resolution shell.
TABLE-US-00002 TABLE 2 Data collection and refinement statistics.
DDB1.DELTA.B-CRBN- DDB1.DELTA.B-CRBN- dBET57-BRD4BD1 dBET70-BRD4BD1
Data collection Space group I 4 2 2 P 65 2 2 Cell dimensions a, b,
c (.ANG.) 313.60, 313.60, 115.779, 115.779, 166.09 593.505 .alpha.,
.beta., .gamma. (.degree.) 90, 90, 90 90 90 120 Resolution (.ANG.)
49.4-6.8 98.8-4.3 (7.0-6.8) (4.4-4.3) R.sub.merge 0.1501 (4.422)
0.4852 (2.183) I/.sigma.I 18.37 (0.77) 8.75 (1.66) Completeness (%)
98.94 (96.59) 99.98 (100.00) Redundancy 26.0 (26.5) 36.9 (36.7)
Refinement Resolution (.ANG.) 6.8 4.3 No. reflections 7315 17739
R.sub.work 0.3351 0.2600 R.sub.free 0.4133 0.3148 No. atoms Protein
1267 1298 Ligand/ion 1 1 Water 0 0 B-factors Protein 276.28 197.19
Ligand/ion 94.22 91.93 Water -- -- R.m.s. deviations Bond lengths
(.ANG.) 0.011 0.008 Bond angles (.degree.) 1.43 1.13 Each dataset
was collected from one crystal. Values in parentheses are for
highest-resolution shell.
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